UNDER CONSTRUCTIONS!!!!!!!!!!!! Coming soon: Methods (step by step with pictures) what we use in the lab including perfusion, immunohistochemistry (pre and postembedding) freeze substitution, high pressure freezing and much more. Stay tuned.

Tissue fixation

A. Perfusion

1. Anaesthetize the animal with Ketylene/Xylene mix.
2. Open the thoracic cavity to access the heart and major vessels. Ensure that the 0.9% NaCl solution is flowing dropwise and that no air bubbles are visible along the tubing.
3. Insert the cannula into the left ventricle and clamp it with a haemostatic clamp
4. Open the right atrium and allow the saline solution to wash out the blood before effective fixation.
5. Clamp the aorta descendens by reaching with haemostatic clamp between the lung and the liver (if we need the brain only)
6. Start perfusion with fixative at a 10mL/min. flow rate for rats, 4mL/min for mice.

B. Fixation

Prior to immunostaining, the tissue has to be adequately fixed to allow a successful immunostaining of antigenic sites. For CNS, optimal fixation is usually obtained by vascular perfusion of the animal with an aldehyde mixture. Aldehydes form crosslinks between proteins in the tissue, thereby protecting them from the rigors of processing and staining techniques.
After dissection of the tissues of interest, tissues can be postfixed. Major advantage of postfixation is to harden the tissues, which facilitates sectioning. However, as certain antigens are very sensitive to fixatives, a considerable decrease in antigenicity may result.

Protocol 1. Zomboni fixative : 0.1M PB
4% paraformaldehyde
15 v/v% picric acid.
See steps1-5 above
6. Perfuse using Zomboni fixative for 30 min. for rats and 15 min. for mice. A total volume of a 300 mL of fixative has to be used for rat and 60 mL for mouse.
7. Dissect out the tissues of interest and immerse directly into PB if post fixation is not required. To postfix tissues, immerse into fixative for an additional 1hr30min on shaker.

Protocol 2. Combined fixative (with Acrolein): 0.1M PB
2% acrolein prepare fresh
4% paraformaldehyde
See steps1-5 above
6. Perfuse with acrolein solution for 5 min. at a 10mL/min. flow rate for rats, 4mL/min for mice.
7. Continue perfusion with paraformaldehyde solution for 30 min. for rats and 15 min. for mice.
8. Dissect out the tissues of interest and immerse directly into PB if post fixation is not required. To postfix tissues, immerse into fixative for an additional 1hr30min.

Preparation of fixatives

Paraformaldehyde dissolves in water at 60ºC at basic pH (add low amount of NaOH to help dissolution).
Picric acid is available as a saturated solution, therefore it has to be filtered to avoid crystals in the fixation solution.
Check pH of fixative, which should be between 7.2 and 7.5. If necessary adjust pH with HCl or NaOH.

Combined fixative for GABA and peptide co-immunolabeling

I. solution : 2 % paraformaldehyde in 0.1 M Na acetate buffer.
PH=6.5

II. solution : 2 % paraformaldehyde in 0.1 M Borate buffer
PH=8.5 0.1% glutaraldehyde

0.2 M Na-acetate buffer 100 ml 1.64 g Na-acetate
pH=6.5 adjust the pH with cc acetic acid

0.2 M Borate buffer 1 l 76.28 g Na-tetraborate-10-hidrate
pH=8.5 adjust the pH with boric acid

For the perfusion: ~ 25 ml I. solution for 2 min
500 ml II. solution for 1 hour
Leave the brain in the skull till next day.

Immunohistochemistry

Preembedding immunoreactions

A. Incubation with primary antibody

1. Wash out sections by rinsing 3x 10 min. with PB 0.1M, then 2x 10 min. with TBS 0.05M
2. Block the sections by incubating 45 min. in 1 mL blocking solution (TBS-5% NGS-0.5% TritonX100 for LM only) for vials, 0.5 mL for 24 wells plates at RT.
3. Use primary antibody diluted into TBS-0.5%NGS-0.1%Azide, incubate 2 days at 4°C to allow antibodies to penetrate into the sections.

B. Incubation with secondary antibody

Protocol 1. Fluorescent staining

1. Wash out primary antibody by rinsing sections 3x 15 min. in TBS.
2. Incubate 5-6 hrs RT with secondary antibody Alexa (A488) diluted 1:500 in TBS in dark.
3. Wash 3x 15 min. with TBS
4. Mount on slides, allow sections to air-dry
5. Add anti-fading reagent Mowiol (has to be at room temperature) and coverslip
6. Seal with nail polish
7. Store at 4°C for temporary storage, at -20°C for long-term storage.

Protocol 2. Avidin-Biotin Complex staining (ABC)

1. Wash out primary antibody by rinsing sections 3x 15 min. into TBS.
2. Incubate 1hr30min. RT with biotinylated secondary antibody diluted 1:1000 in TBS.
3. Wash 3x 15 min. with TBS
4. While washing, prepare ABC solution by diluting 1mL of solution A and 1mL of solution B into 1 mL TBS (1:1000). Let sit RT for 30 min.
5. Add 1 mL of ABC solution to vials and 0.5 mL to the 24 wells plates and incubate 1hr30min RT to allow ABC solution penetrate the samples.
6. Wash 3x 15 min. with TBS
7. Prepare the DAB-Ni solution :
100 mL 0.01M PB
+ 28-35mg DAB N toxic and carcinogen, decontaminate with bleach
R light sensitive, cover with aluminum foil
+ 40mg NH4Cl
+ 5mL 0.05M NiNH4SO4 added drop wise under agitation.
Filter DAB-Ni solution
8. Remove last wash, add 1 mL of DAB-Ni solution to vials, 0.5 mL to 24 wells plates at RT to the samples
9. Allow the DAB solution to penetrate sections 20 min. RT (in dark)
10. Add 10 mL of H2O2 solution (made up by diluting 10mL of 30% H2O2 solution into 10 mL dd H2O) to start the reaction, which will give the black chromogen product. Reaction can take 5-40 min. to develop.
11. Once coloration has appeared, interrupt reaction by removing solution
12. Rinse sections 3x in TBS
13. Mount on slides.

Preparation of buffers

Phosphate buffer 0.2M (PB) pH 7.4
Stock solution A: 0.2M NaH2PO4
Stock solution B: 0.2M Na2HPO4
Add solution A to solution B in a 1:4 ratio until pH reaches 7.4 to give 0.2M PB
Keep at 4°C

Tris-buffered Saline (TBS) 0.1M
Trizma Base 0.1M
Trizma Acid 0.1M
0.9% NaCl

Postembedding immunostainings

Postembedding GABA reaction - EM GABA-gold

Requires high glutaraldehyde (0.5-2%) fixation. Reembedd the area of interest, and cut ultra thin sections. Use Ni or gold grids instead of copper.
Solutions has to be filtered except the primary and secondary antibodies (but filter the buffers what you use to dilute them). The reaction will be carried on droplets on parafilms placed in Petri dishes.

- 1% periodic acid 10’
- dw. 3x3’
- 2% sodium periodate 10’
- dw 3x3’
- TBS (pH=7.4) 3’
- 1% ovalbumin in TBS 30’, for blocking, in moisture chamber
- TBS 2x10’
- Primary antibody 90’ (GABA in TBS containing
1% NGS)
- TBS 2x10’
- 1% BSA+0.5% Tween 20 in TB * 10’ (TB pH=7.4,) Filter the solution twice
- Secondary antibody 1:20 EM-GAR-gold 90’ (in the previous solution*,
moisture chamber)
- dw 3x5’
- 10% uranyl acetat in dw 20’
- dw dip in 4 times
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Counterstain with Pb citrate

Chemicals
Millipore filter: COSTAR r, mStar LB-tm, 0.22mm-es, Cat. no. 8110
Millipore filter paper: Millipore corporation, 0.22 mm-es, GS type, Cat. no. GSWP 013 00
GABA:
EM GAR G15: AuroProbe TM, 15 nm gold labeled goat anti rabbit, Amersham, RPN 422
BSA: Bovine serum albumin: SIGMA, A-3425
Tween 20: Polyoxyethylenesorbitan monolaurate, SIGMA, P-1379
Uranylacetat-dihydrat: MERCK, K18237473, M=424.15