Equipment and reagents
• Two 21-gauge needles
• 2% paraformaldehyde (see Protocol 3, but use 2 gin 100 ml)a
• Imidazole (Sigma): 0.34 g imidazole dissolved in 500 ml PBS
• Quench solution for endogenous peroxidases: 50 ml of 0.1 M phosphate buffer (see Protocol 6), 50 jjlI of 1 M sodium azide (BDH), and 0,09 g glucose (BDH); wann to 37 X and add 20 jxl glucose oxidase (Sigma) just before use
• Avidin/biotin block (Vector Laboratories)
• 0.1% cresyl violet acetatec (Raymond A. Lamb): 1 g cresyl violet acetate in 1000 ml dH,0, heat to boiling point and cool while stirring continuously; filter the cooled liquid in a 0.22 |xm filter
• ABC reagent (Vector Laboratories)
A Immunofluorescent labelling
1 Thaw the slide mounted with frozen tissue for 30-60 min.d
2 Fix the fresh sections by incubating with 2% paraformaldehyde on ice for 10 min.
3 Wash and permeabilize the slide by treating with PBS containing 0.1% Triton for 5 min.
4 Repeat step 3 three times.
5 Quench* the slide with the quench solution for 15 min at 37 °C, G Wash the slide three times with PBS for 5 min each time.f
7 Block the tissue by incubating with 2-5% normal serum for 30 min at room temperature.
8 Dilute the primary antibody to the appropriate concentration in normal serum.
9 Add the antibody to the slide and incubate for 60 min at room temperature.
10 Remove the primary antibody as wash the slide with PBS (see step 6).
11 Add the secondary antibody, diluted to manufacturer's specifications, and incubate for 30 min.
12 Remove the secondary antibody and wash the slide with PBS (see step 6).
13 Incubate the slide with the ABC reagent for 30 min.
14 Remove the secondary antibody as wash the slide with PBS (see step 6).
15 Stain with DAB (see part B).g
Protocol 8 continued
B Diamlnobenzidine detection
1 Place the imidazole into the beaker and draw 20 ml of the solution into a syringe.
2 Push the two needles into the rubber stopper of the DAB vial.
3 Inject the imidazole into the vial through one needle and mix the two compounds thoroughly.
4 Pipette 6 (xlH202 into beaker.
5 Withdraw the DAB solution from the vial using the syringe.
6 Inject the DAB solution through the 0.45 ^m filter into beaker containing H307.
7 Remove the coverplates from the slides and lay slides on racks in trays, which are all stacked up on one side of the vented table and kept solely for this purpose.
8 Cover the tissue with DAB and monitor by eye for a reaction using the negative control to assess colour.
9 After the desired reaction has taken pLace, remove slides from rack and place in a staining rack in a dish of PBS to stop further colour development.
10 When all slides have been reacted, wash each slide twice with PBS, then once with distilled water.
11 Dip the slides a couple of times in cresyl violet acetate to counterstain.
12 Wash the slides with distilled water (see part B, step 10),
13 Dehydrate the tissue and place a coverslip over the tissue. 3 Other fixatives may be used such as acetone and methanol, b DAB is a carcinogen and should be treated with respect. It is most dangerous in powder form and should not be removed from the container.
c Methyl green may be used as a counterstain in place of cresyl violet.
d With perfused tissue sections proceed directly to step 4.
e Quenching is often carried out using 1% H202 in methanol, but we have found best results using glucose/glucose oxidase.
fThe slide may be blocked in avidin and biotin following this step by incubating it with avidin for 15 min, followed by three washes with PBS, then incubate with biotin for 15 min, followed by another three washes with PBS,
BThe slide may be stained with alkaline phosphatase-conjugated secondary antibodies in place of DAB detection. For information, see ref. 10.
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