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DAY ONE
Paraffin sections:
1) Deparaffinize: Xylene , 3x, 5 min each
2) Rehydrate: 100% EtOH, 2x, 5 min each
95% EtOH, 2x, 5 min each
80% EtOH, 1x, 5 min
50% EtOH, 1x, 5 min
Proceed to Antigen Retrieval below (step 3)
Cryo sections (starting with slides that have been baked @ 50°C, 30min):
1) Fix in 4% PFA/PBS, 15min
2) rinse with PBS, 3x, 5 min each
Proceed to Antigen Retrieval below (step 3)
3) Antigen Retrieval: [Vector Labs: Antigen Unmasking Solution #H3300]
For 200 mL: 200mL dH2O
2 mL unmasking solution
a) Fill a rectangular dish 1/3 full with dH20. This will be used as a water
bath for the glass jar containing the diluted unmasking solution. Place
glass jar containing above solution in the bath and heat in microwave
until unmasking solution is visibly boiling – about 5-8 min. (I usually
start preparing this when I’m towards the end of the rehydration steps
outlined above)
b) Place slides from step 2 into hot unmasking solution. Cover with lid.
Transfer entire jar from water bath to bench top. Leave covered for 5 min
at RT.
c) Cool at RT, 5 min with glass lid off.
d) rinse in dH2O, 2x, 5 min each
4) Quench: 13.3 mL 30% H2O2
186.7 mL PBS
200 mL PBS/ 2% H2O2
a) quench 1 min
b) rinse in PBS, 5 min
IMMUNOHISTOCHEMISTRY on PARAFFIN OR CRYOSECTIONS
USING VECTASTAIN ABC ELITE KIT:
5) Block:
a) Block I: 100 mL PBS
1 g Bovine Albumin (Sigma A-9418)
PBS + 1% BSA = PBA
1 hr, RT
b) Block II: PBA
2% serum from animal used to produce 2° antibody (provided
by Vectastain Kit, Vector Labs)
150 μl per slide
30 min, RT
6) 1° Antibody:
a) dilution of choice in PBA
b) humidity chamber, 4°C, overnight
DAY TWO
1) rinse slides in PBS, 2x, 5 min each
2) 2° Antibody: [biotinylated, VectaStain kit]
a) 1:200 dilution in PBA (ex. 5μl antibody per 1000μl PBA)
b) humidity chamber, RT, 1 hr
*MEANWHILE* make avidin-biotinylated peroxidase conjugate:
5 mL PBS
2 drops Reagent A. mix well
2 drops Reagent B. mix well
VectaStain Elite ABC Reagent
Let sit at RT, 30 min before use
c) rinse slides in PBS, 2x, 5 min each
3) VectaStain Conjugation:
a) 30 min, RT
IMMUNOHISTOCHEMISTRY on PARAFFIN OR CRYOSECTIONS
USING VECTASTAIN ABC ELITE KIT:
b) rinse in PBS, 2x, 5 min each
4) Visualization: DAB Substrate Kit for Peroxidase [VectorLabs #SK4100]:
5 mL dH2O
2 drops DAB substrate buffer. Mix well
4 drops DAB. Mix well
2 drops H2O2. Mix well
2 drops nickel-cobalt solution. Mix well
a) use immediately upon making solution, 2-10 min usually enough to stain
b) stop reaction by rinsing with dH2O, 5 min
c) counterstain if desired
5) Dehydrate: 95% EtOH, 2x, 2 min each
100% EtOH, 2x, 2 min each
Toluene, 2x, 2 min each
6) Mount in Permount. Dry overnight
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Immunohistochemistry Pro