Immunohistochemical experiments on mouse tissue sections
1. Bake the paraffin sections in a 60°C oven overnight.
2. Dewax in xylene, add gradient alcohol into water (anhydrous ethanol, 95% alcohol), soak in distilled water for later use.
3. Antigen retrieval
Take 500ml of EDTA antigen retrieval working solution in a 1000ml beaker and heat it on a low-power electric furnace until it boils slightly (in order to prevent peeling). Slowly place the tissue section into the beaker. Continue heating, keeping the liquid at a slight boil for 20 minutes. Remove the beaker from the fire source, take out the slices after natural cooling at room temperature, wash with distilled water once for 3 minutes, and wash with TBS twice for 3 minutes each time.
4. Add one drop or 50ul of 3% hydrogen peroxide solution to each section and incubate at room temperature for 10 minutes to block the activity of endogenous peroxidase. TBS rinsed 3 times, 3 min each time.
5. Remove the TBS solution, add one drop or 50ul of primary antibody (polyclonal antibody obtained from inactivated PLB immunized mice, 1:3000 dilution), normal serum as the negative control, incubate at room temperature for 2 hours, or overnight at 4°C.
6. Wash 3 times with TBS, 5 min each time, remove the TBS solution, add one drop or 50ul polymer enhancer (Reagent A) to each section, incubate at room temperature for 20 min, and rinse with TBS 3 times, 3 min each time.
7. Remove the TBS solution, add one drop or 50ul of peroxidase-labeled anti-mouse/rabbit polymer (reagent B) to each section, incubate at room temperature for 30 minutes, and wash with TBS 3 times, 3 minutes each time.
8. Remove the TBS solution, add a drop or 50ul of freshly prepared DAB to each slice, and develop color for 3-10min.
9. Rinse with tap water, counterstain with hematoxylin for 10 min, and rinse with water. Differentiate with 0.1% hydrochloric acid, rinse with tap water, and turn blue with TBS.
10. No need to dehydrate, directly seal with neutral resin.
Beaker Glass,Beaker 1000ml,Laboratory Beaker 100ml Borocilicate
Post time: Jan-28-2022
