[page 23↓]

2. Materials and Methods

2.1. Cloning

2.1.1. Selection of sequences

Selection of suitable sequences of the amino acid order is necessary to produce highly specific antibodies to investigated channel proteins. I gratefully appreciate the cDNAs of Kir2.1 to Kir2.4 supplied to me by Wischmeyer (2.1), T. Falk (2.3), Kurachi (2.2) and C. Töpert (2.4).

Table 2: vectors

 

Kir2.1

Kir2.2

Kir2.3

Kir2.4

accession code

L48490

X78461

X87635

AJ003065

species

rat

rat

rat

rat

vector of original DNA

pSVSport 1

Bluescript(SK–)

Bluescript(SK+)

pSVSport

number of amino acids

427

427

445

434

The suitable sequences were determined by comparing the homologies between the potassium channels of the Kir2 subfamily (Fig. 7). It was shown by use of the program DNASIS that the greatest difference in amino acid sequence exists at the carboxy-terminal fragments. Consequently, selection of this region is most promising for raising subtype specific antibodies. Selected 50-70 amino acids (Fig. 7) were subcloned into two different vectors. The first vector was used to produce the fusion protein for rabbit immunization and subsequent antibody production, whereas the second vector was utilized for antibody purification (see table 3).

MacDNASIS 03.02 - Hitachi Software Engineering America, USA

vectors:
pGEX-4T-1 - Pharmacia Biotech, Sweden

pQE-40, pet32b - Qiagen, Hilden


[page 24↓]

Table 3: Primer and restriction enzymes (restriction sites underlined)

 

Kir2.1

Kir2.2

Kir2.3

Kir2.4

amino acid sequence

376-427

372-421

372-439

395-434

sequence 5´-Primer

TAG GAT CCC AAA GAG GAA GAG GAC AGT

(27-mer)

CA TGC GGT ACC CTA GCC TTC CTG AGC

(26-mer)

CA TGC GGT ACC TTG GCC CTC ATG AGC

(26-mer)

TGT CAT CTC GAG GAA GGG GTG TCA

(24-mer)

sequence 3´-Primer

CGC GCT CGA GTC ATA TCT CCG ATT CTC

(27-mer)

TA ATT AAG CTT GTA GGG CCG CTC AAG

(26-mer)

TA ATT AAG CCT GTA AGA AAT GTT GTC

(26-mer)

AGA ATG GAT CCG CGC TGA GCT GCT

(24-mer)

expression vector

    

for immunization

pGEX-4T-1

pQE40

pQE40

pGEX-4T-1

for affinity purification

pet32b

pet32b

pet32b

 

restriction sites

BamHI

XhoI

Asp718

HindIII

Asp718

HindIII

XhoI

BamHI

2.1.2. Polymerase chain reaction (PCR)

Polymerase chain reaction is a process of DNA amplification, where the nucleic acid helix is heated and melted into single strands. At appropriate annealing temperatures the added primers hybridize to their respective strand and act as the starting point for DNA polymerase. Multiple repetitions of this process generate amplification of the selected Kir2 DNA sequences.

PCR mixture for 100 µl:

PCR cycle:

 

3 µl plasmid DNA (0.5 µg)

1. Denaturation

45 sec at 95°C

1.5 µl 5´ primer (150 µM)

2. Hot start

addition of polymerase

1.5 µl 3´ primer (150 µM)

3. Primer binding

45 sec at 61°C

1.5 µl dNTP mix (25 mM)

4. Strand synthesis

20 sec at 72°C

10 µl 10x PCR buffer

5. Stop

45 sec at 95°C

80.5 µl H20

6. Repetition from 3.

(25 cycles)

 

7. End

60 sec at 56°C

+ 2 µl Pfu-polymerase (2.5 U)

 

10 min at 72°C

   

The PCR product was cleaned with the “Qiagen PCR Purification Kit”. The successful amplification was further controlled by using electrophoresis on agarose gels and sequenced for confirmation of the correct nucleic acid sequence.

Thermocycler, Omn-E - Hybaid, USA

Pfu-polymerase, 10x PCR buffer - Stratagene, USA

dNTP mix(2´-Desoxyribonucleotid-5´-triphosphat, A,C,T,G) - Boehringer, Mannheim

Primer - MWG-Biotech, Ebersberg


[page 25↓]

2.1.3.  Restriction

Addition of corresponding restriction enzymes to the DNA (vector and insert) results in its being cut at the available restriction sites (see table 3). Therefore, compatible “sticky ends” were generated in both vector and PCR product that become connected in the subsequent ligation. Restriction was performed at 37 °C for 90 minutes and followed by purification of DNA with “Qiagen PCR Purification Kit” / user’s manual.

HindIII, Asp, BamHI - MBI Fermentas (Latvia), Boehringer, Mannheim

2.1.4. Agarose gel electrophoresis

To control the successful restriction, the PCR products were separated in 2 % and the uncut vector in 8 % agarose gel (with 0.5 µg/ml ethidium bromide) in 1x TAE. Electrophoresis was run at 100 V. During this process ethidium bromide intercalates into the DNA and thus, DNA bands can be visualized due to fluorescence under ultraviolet light.

1 x TAE:40 mM Tris acetate, 1mM Na2EDTA, pH 7.6 in H2O

Agarose - Biozym, Hess.Oldendorf

Ethidium bromide - Sigma, USA

Marker - MBI Fermentas, Latvia

6 x Sample buffer - MBI Fermentas, Latvia

Electrophoresis chamber - Kreutz Laborgeräte, Reiskirchen

Power supply unit, Power Pac 200 - Bio-Rad, USA

2.1.5. Determination of DNA concentration

Concentration of DNA was calculated from the optic density OD260nm, measured against water. Double stranded DNA of a 50 µg/ml concentration has a value of 1. The quotient from OD260nm to OD280nm indicates the purity of the DNA. Values greater than 1.8 are indicative of highly purified DNA.

UV-Photometer UV-1202 - Shimadzu, Japan


[page 26↓]

2.1.6.  Ligation

Ligation was performed overnight at 15 °C mixing vector DNA and insert DNA. T4-DNA ligase was then inactivated by incubation at 65 °C for 10 minutes.

Ligation mixture:

10 µl

vector DNA

12 µl

insert DNA

3 µl

10 x ligase buffer - MBI Fermentas, Latvia

5 µl

T4-DNA ligase (1 U/µl) - MBI Fermentas, Latvia

filled up with Millipore H2O to 30 µl

2.1.7. Generation of competent cells

20 ml LB culture medium were incubated with competent bacteria. During the phase of logarithmical growth – at OD600nm between 0.4 and 0.6 at 37 °C on a shaker – cells were extracted and centrifuged for 10 minutes at 5000 x g and 4 °C. The pellets were resolved in 20 ml ice-cold and autoclaved CaCl2 solution (70 mM CaCl2, 10 mM Tris, pH 8.0) and placed on ice for an hour. After new centrifugation for 10 minutes at 5000 x g and 4 °C bacteria were resolved in 2 x 1 ml CaCl2 solution and stored on ice. Finally, bacteria were frozen in liquid nitrogen and stored at -80 °C.

LB-Medium - Gibco Brl, Life Technologies, GB

Centrifuge - Beckman, USA

2.1.8. Transformation

A mixture of 100 µl of competent cells with 20 µl ligated DNA was incubated on ice for 20 minutes under repeated careful shaking, followed by heating in water at 42 °C for 90 seconds and cooling down for 2 minutes on ice. This procedure led to transformation of DNA into competent cells. Afterwards 1 ml LB culture medium was added and the mixture was shaken for 45 minutes at 37 °C. The transformed cells were crossed out on LB Agar (50 µg/ml Ampicillin) in dilutions from 100-10 µl, overnight at 37 °C.

LB-Agar - Gibco Brl, Life Technologies, GB

Ampicillin sodium - Grünenthal, Stolberg[page 27↓] Preparation of plasmid DNA

Individual grown clones on the agar plate were selected and each brought into 4 ml LB culture medium (100 µg/ml Ampicillin) and incubated at 37 °C over night. Purification was performed with the “Qiagen Qiaprep SpinPlasmid Kit” (principle of alkaline lysis). Afterwards correctness of transformation was controlled with restriction of the plasmid (same procedure as after ligation) and separation in agarose gel electrophoresis.

2.2. Protein expression and purification

2.2.1. Overexpression of fusion proteins

Successfully transformed clones grew in 6 ml LB each (+ampicillin) at 37 °C overnight on the shaker (230 rpm), were brought into 500 ml LB culture medium and matured to an OD600 of 0.5-1. The protein expression was induced by adding 1 mM final concentration IPTG (Isopropyl-β-D-thiogalactosid; Roth, Karlsruhe) and subsequent shaking for 4 hours. Finally, cells were harvested with the centrifuge at 5000 g for 20 minutes at 4 °C.

2.2.2. Protein purification

The pellet of expressed protein was resolved in 1 x PBS (for pGEX vector) or lysis buffer (for PQE-40 and pet32b vector) and lysed by sonification and addition of Trition X-100 (only pGEX vector) to a final concentration of 1 % while being shaken for 60 minutes. Afterwards cell de­bris was precipitated by centrifugation at 10000 x g for 30 minutes. Glu­ta­thio­ne-Sepharose 4b (for pGEX vector) or Ni-NTA solution (for PQE-40 and pet32b vectors) was ad­ded to the supernatant containing the solved proteins. The suspension was incubated at room temperature for 60 minutes and then was­hed 5 times in PBS on a column. Eventually the proteins were eluted 5 times with 500 µl glutathione buffer (pGEX vector) or elution buffer (PQE-40 and pet32b vectors). The proteins were dialysed against PBS at 4° C overnight.

Lysis buffer:

NaH2PO4, pH 8

50 mM

Glutathione buffer:

 
 

NaCl

300 mM

Glutathione

10 mM

 

Imidazol

10 mM

Tris-HCl, pH 8

50 mM


[page 28↓]

Washing buffer = Lysis buffer with 20 mM Imidazol

Elution buffer = Lysis buffer with 250 mM Imidazol

Glutathione-Sepharose 4B - Pharmacia, Sweden

Ni-NTA - Qiagen, Hilden

2.2.3. SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)

The method of Laemmli (1970) was used for electrophoretical separation of proteins on 10 % or 12 % SDS-PAGE polyacrylamid gels. The probes were dissolved in 4 x Laemmli buffer and heated at 95 °C for 5 minutes. Gels were loaded with the probes and a marker (Fig. 8). Separation of proteins was performed at 100 V in the stacking gel and at 200 V in the separation gel in the gel chamber filled with 1 x running buffer (0,3 % Tris/HCl, 1.44 % glycine, 0.1 % SDS).

 

12% separation gel

4% stacking gel

30% Acrylamid

3.88 ml

810 µl

2% Bisacrylamid

1.8 ml

375 µl

5x separation/collection gel buffer

2 ml separation gel buffer

(Tris 1.88 M, SDS 0.5%, pH 8.8)

1 ml collection gel buffer

(Tris 630 mM, SDS 0.5%, pH 6.8)

H2O

2.32 ml

2.815 ml

APS (10%)

50 µl

25 µl

TEMED

10 µl

5 µl

4 x Laemmli buffer:

10 % (w/v) ß-Mercaptoethanol

 

4 % (w/v) SDS

 

2 % (w/v) Bromphenolblau

 

20 % (w/v) Glycerol

 

250 mM Tris-HCl, pH 6.8

Electrophoresis chamber Mini-Protean II - Bio-Rad, USA

LMW Standard - Pharmacia Biotech, Sweden

Power supply unit, Power Pac 200 - Bio-Rad, USA

Rainbow protein molecular weight marker - Amersham Life Science, UK

TEMED (N,N,N´,N´,-Tetramethylethylendiamin) - Bio-Rad, USA


[page 29↓]

Visualization of protein bands was done by heating the gels in Coomassie Blue for 10 minutes in the microwave and subsequent decolorization by heating in water several times until the bands demarcate.

Coomassie Blue:

Serva Blue G-250

0.025%

 

Acetic acid

10%

 

Ethanol

50%

2.2.4. Preparative electrophoresis

Electro elution was used for the exact purification of the proteins. Thus, protein was separated in a big preparative SDS gel and stained with Coomassie. The visualized bands were cut with a scalpel and brought into an electro elution chamber. The proteins were eluted at 100 V overnight in elution buffer (25 mM Tris, 192 mM glycine, 0.025% SDS).

Electro elution chamber, S&S Biotrap - Schleicher&Schüll, Dassel

Maxigel chamber, Protean IIxi Cell - Bio-Rad, USA

2.2.5. Determination of protein content

2.2.5.1. BCA (Bicinchoninic acid) assay

40 µl of each protein probe was mixed with 400 µl BCA solution (BCA solution A:B = 50:1) and incubated for 30 minutes at 60 °C. The same procedure was used with a standard row of BSA (bovine serum albumine, 0-200 µg/ml) in PBS. Afterwards extinctions were measured at 550 nm and the protein concentrations calculated from the standard values.

BCA solution A:

BCA-Dinatrium

25.8 mM

 

Na2CO3

160 mM

 

Na2-Tartrat

7 mM

 

NaOH

100 mM

 

NaHCO3

113 mM

BCA solution B:

CuSO4x5H2O

160 mM

2.2.5.2. Bradford assay

In a manner similar to that used with the BCA assay, a standard row and the protein solution were solved in 800 µl probe buffer and subsequently mixed with 200 µl 5 x [page 30↓]staining solution (Rotiquant). After 10 minutes the probes were measured at 595 nm in a photometer and the protein concentration calculated from the standard values.

2.3. Raising of antibodies in rabbits

2.3.1. Immunization and blood taking

For each fusion protein two 4-5 months old “New Zealand white” rabbits were immunized with either 90 or 180 µg of the produced protein. Therefore, 500 µl or 1000 µl antigen were injected intracutaneously after shaving the injection sites. To increase the immune reaction the protein solutions was mixed with an adjuvant and two booster immunizations were performed after an interval of 3-4 weeks.

Immunization solution:

Complete Freund’s adjuvant (CFA)

1.25 ml

Sigma, USA

Antigen (360 µg) in PBS

750 µl

 

Before the first injection a blood sample was taken (preimmune serum) and starting with day 35, 30 ml blood were taken weekly from the rabbit ear. The blood coagulated overnight at 4 °C and the separated serum was centrifuged at 2500 x g. The complement system was inactivated by heating for 1 hour at 56 °C and the sample was stored at –80°C. After 12 weeks the animals were killed by cervical dislocation and exsanguinated, the blood was processed as described above.

2.3.2. Determination of antibody titer, ELISA

Determination of antibody titer is mandatory to observe and follow the production of antibodies. The “enzyme-linked immunosorbent assay” (ELISA) is an adequate method. Micro titer plates are coated with antigen and subsequently incubated with the antibody whose concentration should be determined. A secondary antibody is added onto the plates after the washing process, and a staining reaction visualizes the amount of primary antibody.


[page 31↓]

Table 4: ELISA

Procedure:

   

Antigen coating

100 µl

1 µg/ml in coating buffer1

15 h

Saturation of unspecific bindings

150 µl

saturation solution2 pH 7.2

1 h

Primary antibody (e.g. serum)

100 µl

diluted in saturation solution

2 h

Washing (3x)

200 µl

PBS

 

Secondary antibody

100 µl

peroxidase-conjugated AB goat-anti-rabbit, 1:1000 in 1mg/ml PBS-Hb

 

Washing (3x)

200 µl

PBS

 

Visualization

100 µl

incubation solution3 pH 4.2

5 min

Stop

100 µl

block solution (0.1% sodium azide)

 

Measurement of extinctions at 450 nm

   

1

50 mM

Na2CO3 in water, pH 9.6

  

2

50 mM

NaH2PO4xH2O

350 mM

NaH2PO4xH2O

 

150 mM

NaCl

100 mM

NaCH3CO2x3H2O

 

0.05%

Tween 20

1 mg/ml

ABTS

 

0.02%

Sodium azide

0.88 mM

H2O2

 

1 mg/ml

Hemoglobin

  

ELISA-Micro plate, Falcon3912 - Becton Dickinson, USA

ELISA Photometer, Dynatech MR 5000 - Dynatech Laboratories, USA

P-GaR (goat anti rabbit antibody) - Vector, USA

2.3.3. Competitive ELISA

This special modification of ELISA is preceded by preincubation of the antibodies for 2 hours with different antigens in varying concentrations (10 µg – 0.1 ng). The results indicate both the affinity of antibodies (decrease of extinctions, see Fig. 12) and the absent cross reactivity to other antigens.

2.4. Purification of antibodies

2.4.1. Removal of IgM

For all experiments only antibodies of the immunglobuline class G (IgG) were used, because only this class can be detected with the secondary antibodies used. Furthermore, included antibodies of the IgM class would cause undesirable background in the immunohistochemical experiments. Therefore, IgM antibodies were removed using a Superdex-200 column: 2.5 ml rabbit serum from the fractions with the highest antibody [page 32↓]titer were pumped through the column. The bigger IgM molecules move faster through the porous agarose beads than the smaller IgG antibodies. The resulting fractions with the highest titer were detected due to their extinctions (Fig. 9) and pooled (circa 10 x 2 ml).

Fraction collector FC 203 B - Abimed Gilson, USA

Peristalsis pump Minipuls 3 - Abimed Gilson, USA

Photometer 112, UV/VIS Detector - Abimed Gilson, USA

Column Superdex 200 prep grade - Pharmacia Biotech, Sweden

Writer BD 111 - Kipp & Zonen / Sci-Tec Instruments, Canada

2.4.2. Removal of cross reactivity

The IgM-purified serum was tested for cross reactivity to the fusion part of the immunization protein, to bacterial surfaces and to other Kir channels. In case of cross reactivity, the serum was incubated with the detected undesirable antigens.

The antibodies to the fusion part1 and to bacterial structures were removed by applying a bacterial suspension (expression of bacteria with only the fusion part, centrifugation for 15 minutes at 4000 g, treatment with 0.5 % formaldehyde and autoclave). The bacterial suspension was added to the 1:100 in PBS-Hb diluted serum, the amount having been determined in previous experiments (Fig. 10), incubated overnight at 4 °C and centrifuged for 15 minutes at 4000 g. The supernatant contains the wanted antibodies.

After determination of cross reactivity to closely related Kir channels, these antibodies were removed. Therefore, nitrocellulose membranes were coated with the cross reacting antigen2 overnight (2-8 µg/ml in PBS, according to previous experiments). Free binding sites were then saturated with 5 % NGS (normal goat serum) in PBS for 3 hours and the membranes incubated with the serum. Thus, the desirable antibodies were able to bind. Finally, the supernatant was checked again for cross reactivity (Fig. 11)

NGS, goat serum - PAN Systems, Aidenbach

Nitrocellulose - Schleicher & Schuell, Dassel


[page 33↓]

2.4.3.  Affinity purification

Affinity purification is performed in a manner similar to that used for removing of cross reactivity by coating nitrocellulose membranes with antigen overnight (2 µg/ml in PBS). Therefore, however, the second fusion protein3 was used which is the Kir channel fragment cloned into the pet32b vector (see table 3). The membranes were washed three times with PBS and unspecific bindings blocked with 5 % NGS in PBS for 1 hour, followed by incubation with the serum (1:100 in 5 % NGS) for 4 hours. The membranes were washed again three times with PBS and eluted for 30 minutes1. Finally, the elution was dialysed against 20 mM phosphate buffer (pH 6) overnight.

1 Elution buffer (pH 2,5 with HCl) = Glycine 200 mM, NaCl 150 mM, BSA 1 mg/ml

2.4.4. Chromatofocussing

Determination of the isoelectric point of antibodies is mandatory for a pH dependent concentration of antibodies. Therefore, an anion exchange column (PBE-94 material) is used which separates different proteins according to their varying charge. The column was equilibrated above the expected isoelectric point with 10 ml loading buffer (25 mM Ethanolamine, pH 9.4) and loaded with 5 ml affinity purified antibody solution that was dialysed against loading buffer. The column was then rinsed with 10 ml loading buffer and eluted with 25 ml Polybuffer (pH 6.0). The elution was collected in 0.5 ml fractions. The pH of the fractions with the highest affinity to the antigen (ELISA) determined the isoelectric point of the antibodies.

PBE 94, Polybuffer exchanger - Pharmacia, Schweden

Polybuffer 96-CH3COOH - Pharmacia, Schweden

2.4.5. Concentration of antibodies

The antibody concentration was increased using a SP-Sepharose cation exchange column. The sulfopropyle groups of the sepharose material bind positively charged proteins above a pH of 2. The column was equilibrated with 20 ml loading buffer (20 mM phosphate buffer, [page 34↓]pH 6.0). The column was then loaded with affinity purified antibodies that are positively charged at pH 6. Finally, 200 µl-fractions were eluted with a basic buffer (200 mM Na2CO3, pH 9.0) by detaching the antibodies due to the negative charge. The fractions were checked by ELISA for antibody titer and the richest were pooled and dialysed.

SP-Sepharose Fast flow - Pharmacia, Schweden

2.5. Characterization of antibodies

2.5.1. Analysis of specificity in Western Blots

2.5.1.1. Preparation of brain homogenates

Brains were rapidly dissected from deeply anesthetized (ether) adult Wistar rats (5-9 weeks) and rapidly frozen on dry ice. 2 ml of homogenization buffer were added per gram of rat brain and the solution was homogenized 10 times in a Dounce homogenizer. The ho­mo­ge­na­te was centrifuged at 4 °C for 10 minutes at 600 x g to re­mo­ve cell de­bris and nuclei, and the supernatant wa­s cen­tri­fu­ged at 100.000 x g for 10 minutes to pel­let mem­bra­nes. The pellet which contains cell membranes and organelles was resuspended in homogenization buffer.

Dounce homogenizer - Wheaton, USA

Homogenization buffer

 

added before use:

 
 

Saccharose

250 mM

 

Aprotinin

2 µg/ml

 

Hepes; pH 7.4

4 mM

 

Pepstatin A

1 µg/ml

 

EDTA

1 mM

 

Leupeptin

1 µg/ml

 

Na-azide

5 mM

 

PMSF

0.5 mM

2.5.1.2. Western Blotting

Proteins were separated on an SDS gel and transferred to nitrocellulose membranes using the BioRad semidry blot device which is constructed as follows:

The Whatman paper and the nitrocellulose were previously rinsed in semidry buffer. Blotting was performed at 300 mA / gel for 30 minutes. After transfer, the proteins were visualized on the membrane by using Ponceau S-solution. The nitrocellulose membranes were cut in strips containing single protein lanes and totally decolorized in PBS.

Semidry buffer

 

Ponceau S-solution

 
 

Tris

48 mM

 

trichloric acid

3%

 

Glycine

390 mM

 

Ponceau S

400 mg/l

 

SDS

0.04% (w/v)

   
 

Methanol

20% (v/v)

   

2.5.1.3. Immune detection

The unspecific binding sites at the membranes were saturated using 5 % low-fat milk in PBS containing 0.1 % Tween-20 for 1 hour. The primary antibody solution (anti-Kir2.1 [1:1000], anti-Kir2.2 [1:500], anti-Kir2.3 [1:500], anti-Kir2.4 [1:5000] in 5 % milk/PBS/0.1 % Tween-20) was added and incubated overnight at 4 °C. The blots were washed 5 times in PBS/0.1 % Tween-20 (1 hour in total) and the secondary antibody was added (1:500-1:1000 in PBS/0.1 % Tween-20) for 2-3 hours. Thereafter, the membranes were washed again 5 times in PBS/0.1 % Tween-20 and developed.

2.5.1.4. Visualization by use of alkaline phosphatase (aP)

The aP-conjugated secondary antibody was visualized using the substrate NBT/BCIP (Ni­tro Blue Te­trazol­­i­um / 5-bro­mo-4-chlo­ro-3-in­do­lyl phos­pha­te).

Procedure:

The blots were incubated in 10 ml aP-development solution for 2-25 minutes depending on visualization intensity. The reaction was stopped by washing with PBS.

aP-development solution:

 

Tris; pH 9.5

100 mM

MgCl2

50 mM

NaCl

150 mM

added before use:

 

NBT (75 mg/ml in 700 µl DMF + 300 µl H2O)

50 µl/10 ml

BCIP (50 mg/ml in 1ml DMF)

37.5 µl/10 ml


[page 36↓]

2.5.2.  Analysis of specificity by transfected cells

2.5.2.1. Liposome-mediated transfection

Afri­can green monkey COS-7 kidney cells we­re cultured at 5 % CO2 in Dulbeccos modified Eagle medium supplemented with 10 % fe­tal calf-serum on 6-well-dishes containing four glass plates. At 60 % confluence cells were tran­si­ent­ly transfected using lipofectin-DNA solution and incubated for 6 hours. After adding serum-containing medium, cells were incubated for 48-72 hours at 37 °C.

Production of lipofectin-DNA solution from solution A and B:

Solution A: 1-2 µg DNA diluted in 100 µl serum free medium per transfection

Solution B: 2-10 µl lipofectin were diluted in 100 µl SFM

Both solution were incubated for 30-45 minutes at room temperature, mixed carefully and incubated for another 10-15 minutes at room temperature. The lipofectin-DNA solution was then used for transfection.

lipofectin - Gibco Brl, Life Technologies, GB

2.5.2.2. Detection of transfected cells by immunofluorescence

Cells were washed in PBS followed by fi­xa­tion in 4 % phosphate-buffered formaldehyde and three washing steps with PBS. After blocking for 1 hour at room temperature in 5 % normal goat serum (NGS) in PBS containing 2 % BSA and 0.3 % Triton-X-100, cells were incubated with the primary antibody for 2 h at 37 °C (in 5 % NGS/ 2 % BSA/ 0.3 % Triton). Subsequently, cells were washed 3 times in PBS, incubated with the secondary an­ti­bo­dy (fluorescent Oregon Green 488-goat anti-rabbit IgG 1:200, diluted in PBS/ 2% BSA/ 0.3 % Triton) for 2 hours at room temperature and was­hed again with PBS. Coverslips were mounted with moviol 4-88 containing 2.5 % of 1,4-di­a­zo­bi­cy­clo-[2.2.2]-octane as anti-fading substance and vie­wed using fluorescence microscopy at 488 nm.

Oregon Green 488-goat anti-rabbit IgG 1:200 - Molecular Probes, USA

moviol 4-88 - Hoechst, France

1,4-di­a­zo­bi­cy­clo-[2.2.2]-octane - DABCO; Sigma, Munich


[page 37↓]

2.6.  Immunocytochemistry

2.6.1. Perfusion fixation of rat brains

Adult Wi­star rats were deeply anesthetized with et­her, injected with muscle relaxant and coagulation inhibitor (ketamine, rompun and heparin adapted to body weight) and perfused transcardially with:

Brains were dissected out, embedded in 2 % agarose in sucrose solution, and cut transversely into 2-3 mm thick sections. Brains were cytoprotected in 10 % sucrose for 1 hour and in 30 % sucrose overnight, fro­zen in hexane at –60 °C, attached to cork plates and stored at –80 °C until use.

PGPIC (fixation solution), pH 7.4

sucrose solution, pH 7.4

 

paraformaldehyde

4% (w/v)

 

sucrose

146 mM

 
 

NaH2PO4xH2O

100 mM

 

NaH2PO4xH2O

100 mM

 
 

glutaraldehyde

0.05% (w/v)

   
 

picric acid

0.37%

   

2.6.2. Rat brain slices for light microscopy

For light mi­cro­sco­py, fro­zen brain blocks were cut into 20 µm cryosections on a microtome and rinsed se­ve­ral times in PBS. After treatment with 1 % sodium borohydride in PBS for 15 minutes to remove excessive al­de­hy­des and two washing steps with PBS for 15 minutes, sections were preincubated in 10 % NGS in PBS con­tai­ning 0.3 % Triton-X-100 and 0.05 % phenyl hydrazine to reduce the endogenous pe­ro­xi­da­ses. Sections were washed subsequently three times in PBS, followed by incubation for 36 h at 4 °C in the primary antibody solution (rabbit anti-Kir2.1 [1:400], anti-Kir2.2 [1:100], anti-Kir2.3 [1:100], anti-Kir2.4 [1:5000] in 10 % NGS in PBS containing 0.3 % Triton-X-100, 0.1 % sodium azide and 0.01 % thimerosal). Sec­tions were washed in PBS two times for 20 and 40 min, preincubated for 1 h in PBS-A (2 mg BSA/ ml PBS) and incubated for 16 h at room temperature in the secondary antibody solution (biotinylated goat anti-rabbit IgG 1:2000 in PBS-A containing 0.3 % Triton-X-100 and 0.1 % sodium azide). After two [page 38↓]washing steps in PBS, preincubation for 1 hour in PBS-A and incubation in 1:1000 avi­din-biotin-complex (10 µl Elite A dissolved in 10 ml PBS-A, 10 µl Elite B added) for 6 h at RT, sec­tions were washed three ti­mes in PBS (10, 20 and 30 minutes), followed by a preincubation in DAB-solution (0.5 mg DAB in 1 ml 50 mM Tris-buffer, pH 7.6, containing 10 mM imi­da­zo­le) for 15 min. Sections were visualized by adding am­mo­ni­um nickel sul­fa­te (0.3 % final con­cen­tra­tion) and H2O2 (0.015 % final concentration) for 3 min. Finally, they were was­hed several times in PBS, mounted onto gelatin-coated slides, dried for 30 minutes, dehydrated in increasing alcohol concentrations, brought into xylol, and co­vers­lip­ped with entellan. In control experiments, the primary antibody was preabsorbed with 10 µg specific antigen per 1 ml an­ti­bo­dy solution.

Elite A (avidin) - Vector Laboratories, USA

Elite B (biotinylated peroxidase) - Vector Laboratories, USA

DAB solution (3,3-di­a­min­o­ben­zi­di­ne) - Sigma, Mu­nich

2.6.3. Rat brain slices for fluorescence microscopy

For fluorescence mi­cro­sco­py, fro­zen brain blocks were cut into 20 µm cryosections on a microtome and rinsed in PBS. After treatment with 1 % sodium borohydride in PBS for 15 minutes and two washing steps with PBS for 15 minutes, sections were incubated in 10 % NGS in PBS con­tai­ning 0.3 % Triton-X-100 to saturate unspecific binding sites. Slices were incubated for 36 h at 4 °C in the primary antibody solution (rabbit anti-Kir2.4 [1:5000] in 10 % NGS in PBS containing 0.3 % Triton-X-100, 0.1 % sodium azide and 0.01 % thimerosal). Sec­tions were washed in PBS two times for 20 and 40 min, preincubated for 1 h in PBS-A (2 mg BSA/ ml PBS) and incubated for 16 h at room temperature in the secondary antibody solution (fluorescent anti-rabbit IgG in PBS-A containing 0.3 % Triton-X-100 and 0.1 % sodium azide). Slices were was­hed again 3 times in PBS and, finally, coverslipped with moviol 4-88.

2.6.4. Coating of slides

Glass slides were washed thoroughly (dishwasher), dived into warm gelatine solution for 3 minutes and dried for 2-3 days at room temperature in a dust-free environment.


[page 39↓]

Production of gelatine solution:

15 g gelatine and 1.76 g potassium chromosulfate-12-hydrate were diluted at 70 °C in 630 ml H2O. 300 ml 100 % ethanol and 70 ml acetic acid were then added and the solution filtered.

2.6.5. Cresyl violet staining

Microtome sections were coated on glass slides, dried for 30 minutes and dehydrated in 70 % ethanol overnight. The slides were washed for 2 minutes in H2O and stained in cresyl violet solution for 20-30 minutes. Afterwards, they were washed again for 2 minutes in H2O, dehydrated in increasing ethanol concentrations (70 %, 86 %, 96 %, 100 % I, 100% II for 5 minutes each), brought into xylol, and co­vers­lip­ped with entellan.

Cresyl violet solution

 
 

cresyl violet acetate

0.2% (w/v)

 
 

acetic acid

20 mM

 
 

pH 4.0 with NaOH, always filter before use

 

2.6.6. Electron microscopy

2.6.6.1. Immunoreaction

For elec­tron micros­co­py, fro­zen brain blocks (sto­red at -80 °C) were thawed in 30 % sucrose in PBS. 40 µm slices were sectioned on a vibratome at 4 °C and rinsed several times in PBS. Sections were in­cu­ba­ted with an­ti­bo­dies as in pa­ra­graph 2.6.2., with one exception: Triton-X-100 (0.1 % fi­nal con­cen­tra­tion) was ad­ded only to the 0.05 % phenyl hydrazine preincubation solution. Sections were visualized for 10 min by ad­ding 0.015 % H2O2 but omit­ting nickel ions.

Composition of secondary antibody solution:

biotinylated 2° AB

1:2000

in PBS-A with 0.1% Na-azide

0.8 nm gold-labeled 2° AB

1:40

in PBS with 0.1% BSA-C; 0.1% Tween und 0.1% NaN3

2.6.6.2. Staining with the avidin-biotin method

After two washing steps in PBS and incubation for 1 hour in PBS-A, sec­tions were incubated with the avidin-biotin complex for 6 hours at room temperature. Slices were [page 40↓]washed three ti­mes in PBS (10, 20 and 30 minutes), followed by a preincubation in DAB-solution (0.5 mg DAB in 1 ml 50 mM Tris-buffer, pH 7.6, containing 10 mM imi­da­zo­le) for 10-12 minutes. Sections were visualized by adding H2O2 (0.015 % final concentration) for 3 minutes (without am­mo­ni­um nickel sul­fa­te). After thoroughly washing in PBS, the tissue was embedded in araldite.

2.6.6.3. Gold-silver-enhancement

In the experiments in which a gold-labeled secondary antibody was used, the slices were washed 10, 20 and 30 minutes in PBS-Tween (0.05 % Tween 20), followed by a 15 minutes fixation step in 2 % glutaraldehyde in PBS. After a short rinsing in PBS, the sections were washed 5 times in 150 mM NaNO3 solution and silver-enhanced in the dark for 25 minutes. The sections were washed a few times in PBS and fixed in 2 % glutaraldehyde for 10 minutes, rinsed again in PBS and subjected to the following araldite embedding.

2.6.6.4. Araldite embedding

Slices were post-fixed with 1 % os­mi­um te­tro­xi­de in PBS for 15-30 minutes, washed in phosphate buffer (100 mM NaH2PO4xH2O; pH 7.4 with NaOH), dehydrated through graded ethanol and block stai­ned with 2 % uran­yl ace­ta­te in 70 % EtOH for 10 min at 4 °C. The sections were dehydrated again in graded ethanol (3 x 70 % EtOH, 2 x 95 %, 3 x 100 %), transported into propylene oxide twice for 5 minutes and incubated overnight in the propylene oxide/ araldite mixture. Afterwards, the slices were incubated 2 x 2 hours in the embedding solution and embedded in araldite bet­ween two sheets of Aclar plastic. The polymerization of the araldite was performed for 24-60 hours at 65 °C. Finally, the regions of interest were cut and glued to a carrier plastic.

Ultrathin (60-80 nm) and semithin tissue sections (800-1000 nm) were cut alternately with a diamond knife and the ultrathin sections were collected on copper grids for electron microscopy.

Aclar plastic - Ted Pel­la, USA

Diatome diamond knife

Araldite solution: Araldite and DDSA were intensively mixed in a ratio of 30:24.


[page 41↓]

Propylene oxide / araldite mixture: 55 g araldite solution was mixed with 1.5 ml DMP-30 and 50 ml propylene oxide.

Embedding solution: 100 g araldite solution was intensively mixed with 2 ml DMP30.

2.6.6.5. Contrasting the ultrathin sections

The sections we­re counterstained for 5 minutes with 5 % uran­yl ace­ta­te in water, intensively washed in water, incubated for 5 minutes in le­ad citrate solution and washed again thoroughly in water. Thereafter the sections were examined with a trans­mis­sion electron microscope.

trans­mis­sion electron microscope - EM 900, Zeiss

lead citrate solution

 
 

lead citrate

1.33 g in 50 ml

 
 

sodium citrate

1,76 g auf 50 ml

 
 

pH 12.0 with NaOH

  

2.6.6.6. Toluidin blue staining

The semithin tissue sections were dried on glass slides and stained for 2-5 minutes in toluidin blue solution. They were then rinsed with H2O and and co­vers­lip­ped with entellan.

Toluidin blue solution; pH 9.35

 
 

Na2B4O2 x 10H2O

1% (w/v)

 
 

Toluidin blue

1% (w/v)

 
 

sucrose

40% (w/v)

 

2.6.7. Histological analysis

The nomenclature of Paxinos and Wat­son for the rat brain4 is used throughout this re­port to refer to anatomical structures.


Footnotes and Endnotes

1 Pompéia, C. 1996

2 Veh, R.W. 1995

3 Pitt, J.C. 1998

4 Paxinos, G. and Watson, Ch. 1998



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