Performing Immunohistochemistry on a Non-Human Primate Brain Tissue

Samenvatting

Source: Eid, L. & Parent, M. Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy. J. Vis. Exp. (2017).

This video demonstrates the preparation of aldehyde-fixed non-human primate brain tissue using pre-embedding immunohistochemistry for electron microscopy. It outlines the steps for immunolabeling a specific neuronal protein, followed by a peroxidase reaction that produces a colored precipitate, enabling visualization of the target protein in electron microscopy.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Pre-embedding Immunohistochemistry (Figure 1C)

1. Prepare a 4 L stock solution of Tris-buffered Saline (TBS, 50 mM, pH 7.6) as follows.
   1. Measure 2L of distilled water in a 4 L beaker, add 24.23 g of trihydroxymethyl aminomethane (THAM; C₄H₁₁NO₃), and stir to dissolve.
   2. Adjust the pH to 7.6 with approximately 148 mL of 1 N HCl. The acid should be added with caution to avoid reaching a pH below 7.6. The total volume should be 4 L.
      CAUTION: HCl is highly corrosive. Wear the appropriate PPE.
2. Select sections containing the region of interest to be processed for EM immunohistochemistry.
3. Wash the free-floating sections 3x in PBS (0.1 M, pH 7.4) for 5 min at RT to rinse the antifreeze solution.
4. Prepare a 0.5% solution of NaBH₄ diluted in PBS.
   1. Weigh 0.05 g of NaBH₄ and dilute it in 10 mL of PBS. Do not cover. Prepare this solution just before use.
5. Incubate the sections in the freshly prepared NaBH₄ solution for 30 min at RT. Rock gently. Do not cover.
6. Wash 3x in PBS for 10 min at RT, rocking vigorously until none of the reaction gas remains.
7. Prepare a blocking solution for EM with 2% appropriate normal serum and 0.5% cold fish gelatin diluted in PBS.
   NOTE: The quantity should be calculated in order to have enough for the following three steps. Use a serum made from the same animal species hosting the secondary antibody. Avoid the use of antigen retrieval methods or the addition of small amounts of triton (to increase the penetration of antibodies), as these methods significantly compromise the quality of the tissue.
8. Incubate the sections in the blocking solution for 1 h at RT. Rock gently.
9. Prepare primary antibody solution diluted in the blocking solution.
   NOTE: The concentration of the primary antibody is usually the same as that of LM immunohistochemistry, but consider conducting tests with different antibody concentrations beforehand, as some primary antibodies may not work with acrolein. If so, it is possible to use a mix of glutaraldehyde (0.1 - 2%) and 4% PFA for transcardiac perfusion and obtain similar results. Optimize the incubation time and temperature as well.
10. Incubate the sections in primary antibody solution overnight at RT with gentle rocking. Use a previously tested incubation time and temperature.
11. Wash the sections 3x in PBS for 5 min at RT with gentle rocking.
12. Prepare a 1:1,000 solution of biotinylated secondary antibody diluted in the blocking solution.
    NOTE: The secondary antibody must be raised against the host species used to generate the primary antibody.
13. Incubate the sections in a secondary antibody solution for 1.5 h at RT. Rock gently.
14. Prepare an avidin-biotin-peroxidase (ABC) solution at least 60 min prior to the end of the secondary antibody incubation.
    1. Use a calibrated pipette to measure 8.80 µL/mL of solutions A and B and dilute them in PBS.
    2. Rock mildly for at least 60 min at RT to allow the complete binding between the avidin and biotin molecules.
15. After the incubation in the secondary antibody solution, wash 3x in PBS for 10 min at RT.
16. Incubate the sections in the ABC solution for 1 h at RT. Rock gently.
17. Wash once in PBS and twice in TBS for 10 min at RT with gentle rocking.
18. Prepare a fresh solution of 0.05% 3,3'-diaminobenzidine (DAB) with 0.005% H₂O₂ diluted in TBS.
    1. Weigh 12.5 mg of DAB and dilute it in 25 mL of cold TBS. Protect from light.
       CAUTION: The DAB powder is highly volatile and harmful if inhaled. It is carcinogenic and teratogenic. Thus, pregnant or nursing women should not manipulate this product, even when diluted. Use an N95 mask when manipulating and wear PPE.
    2. Filter the solution and add 4.5 µL of 30% H₂O₂ just before use.
19. Incubate sections in the DAB solution for 3 to 7 min at RT. Rock gently.
    NOTE: The brown precipitate should not be too dark in order to avoid a high level of background staining. The incubation time should be optimized accordingly.
20. Stop the reaction by quickly washing twice in cold TBS, then twice for 10 min in cold TBS at RT, followed by twice for 10 min in PB, with mild rocking.
    NOTE: The use of PB (and not PBS) is critical in order to eliminate any traces of NaCl, as it would react with osmium and form crystals.

Resultaten

Figure 1: Schematics of Essential Steps of the Protocol.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Dibasic anhydrous sodium phosphate	Fisher scientific	S374-500
Monobasic monohydrate sodium phosphate	EM Science	SX0710-1
Sodium chloride (NaCl)	Fisher scientific	S271-3
Hydroxymethyl aminomethane (THAM)	Fisher scientific	T370-500
HCl	EMD	HX0603-3	1N dilution. Product is corrosive. Use with appropriate potection.
NaOH	EMD	SX0590-1	5N dilution. Product is corrosive. Use with appropriate protection.
Paraformaldehyde	Sigma	P6148	4% dilution. Product is highly volatile in its powder form and highly toxic. Use with caution under a venting hood with appropriate protection.
Acrolein (90%)	sigma	110221	3% dilution. Product is highly toxic. Use under a venting hood with appropriate protection.
Autopsy venting table	Mopec	CE400
Electronic perfusion pump	cole parmer	masterflex L/S 7523-90
Needle (perfusion)	terumo	NN-1838R	18G 1 1/2
Needle	terumo	NN-2713R	21G 1/2
Ketamine			20 mg/kg
Xylazine			4 mg/kg
Acepromazine			0.5 mg/kg
Scalpel
Scalpel blades	Feather lance	201011 J9913	No.22 for surgery and No. 11 for EM
Surgical scissors
Rongeurs
Vibratome	Leica	VT 1200S	Calibrate blade before each use, when the device allows it
Vibratome razor blade	Gillette	GIN 642107
Glycerol	Fisher scientific	G33-4	30% dilution
Ethylene glycol	Fisher scientific	E178-4	30% dilution
Sodium borohydride	sigma	S-9125
Normal horse serum	Jackson immunoResearch Laboratories	008-000-121	2% dilution
Cold-fish gelatin	Aurion	900.033	0.5% dilution. Original product is concentrated at 40%
Primary antibody, SERT	Santa Cruz biotechnology	SC-1458	1/500 dilution
Primary antibody, ChAT	Chemicon (Millipore)	AB144P	1/25 dilution
Primary antibody, TH	ImmunoStar	22941	1/1000 dilution
Biotinylated secondary antibody, goat	Vector laboratories	BA-9500	1/1000 dilution
Biotinylated secondary antibody, mouse	Vector laboratories	BA-2000	1/1000 dilution
Vectastain elite ABC kit	Vector laboratories	PK6100	8.8 µL/mL of A and B each
3'3 diaminobenzidine (DAB)	Sigma	D5637	0,05% dilution. Product is highly volatile in its powder form and toxic. Do not throw waste in the sink.
Peroxide 30%	Fisher scientific	H-323	0,005% dilution