Heat-Assisted Antigen Retrieval in Freshly Frozen Brain Tissue

MICROWAVE HEAT-ASSISTED ANTIGEN RETRIEVAL IN FRESHLY FROZEN BRAIN TISSUE 

Fresh rat brain tissue is immediately immersed in 2-methyl butane at -15 to -25°C for several minutes and stored at -70°C. Sections (10 p,m) are cut in the parasagittal plane on a cryostat, which are thaw-mounted onto poly-L-lysine-coated glass slides and stored at -70°C. The sections are dried on a slide warmer at the lowest setting to avoid excessive drying. They are fixed with 4% formaldehyde in PBS (pH 7.5) for 30 min and then rinsed three times for 10 min each in PBS to remove excess fixative. This is followed by treatment with 0.3% hydrogen peroxide in PBS for 30-40 min to quench endogenous peroxidase activity. 

After being washed in three changes of 5 min each in PBS, the slide is placed in a plastic jar containing 60ml of 10 mM sodium citrate buffer (pH 6.0) and 0.04% Triton X-100. The jar is loosely covered with its screw cap and heated for 5 min in a microwave oven at high power. The buffer starts to boil after 90 sec. The heating process is interrupted at intervals of 1 min so that the fluid level can be checked and replenished in the jar. The sections must not be allowed to dry. The jar is removed from the oven and allowed to cool to room temperature for 20-30 min. The sections are rinsed in three changes of 5 min each in PBS. 

MICROWAVE HEAT-ASSISTED RAPID IMMUNOSTAINING OF FROZEN SECTIONS 

Sections (7 jim thick) of freshly frozen tissues are mounted on silane-coated slides and fixed with 4% buffered formaldehyde (pH 7.0) for 20 sec (Richter et ah, 1999). The sections are rinsed in TBS (pH 7.4) for 15 sec, followed by incubation with EPOS antibody for 3 min at 37°C in an incubation chamber. They are rinsed twice for 15 sec each in TBS and then developed with peroxidase-DAB detection kit (Dako) in a microwave oven (500 W) for 1 min during microwaving, the slides are cooled by a cold water bath (Werner et al., 1991). After being rinsed in tap water, the sections are counterstained with hematoxylin for 10 sec. They are rinsed in tap water and cover-slipped. 

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