Bitan: parafin embedded IHC

IMMUNOHISTOCHEMISTRY PROTOCOL FOR PARAFFIN SECTIONS

DAY 1

0 start pressure cooker at 450o -fill plastic green slide container with citrate buffer

1 Deparaffinize by placing slides in 4 changes of xylene (in hood) 4, 2, 2, 2 min -labeled P1-P4 -bring EtOH to hood

2 Rehydrate the slides by placing in 100% alcohol (x2) 2 min each

3 Place in 95% alcohol (x3) 2 min each

4 Place in 70% alcohol 2 min

5 Place in distilled water 2 min

6 Place slides in citrate buffer and set in hot pressure cooker 20 min -can hear it boiling -put grid on bottom (1/2 in water) -line arrow with handle

7 Remove from heat and allow to cool to RT (antigen retrieval) 15-30 min -use ice packs around slide container

8 Quickly rinse/dip in distilled water (x2) 10 sec

9 Transfer slides into a new clear container and rinse with PBS on shaker 3, 5, 5 min -prepare 0.3% Hydrogen Peroxide solution (200 ml/container) 1 ml 30% H2O2 + 100 ml ddH2O -be aware that certain primary antibodies require an additional reduction step with sodium borohydride after H2O2 step (ie: glycine, glutamate). Check data sheet

10 Place in H2O2 solution 30 min -prepare blocking solution: 4 sect/slide, 50uL/sect, 1/3 10% BSA, 0.05% NHS, PBS

11 Rinse in PBS (x2) 3, 5 min -get incubation box, cut 2 kimwipes into stripes, cover spaces, wet with squirt bottle

12 Pap pen slides and return them to PBS for additional rinse 7 min

13 Rinse in PBST 7 min -0.1% Tween

14 Apply Blocking Solution to slides inside incubation boxes 45 min @ 3¬¬7o -add 45 uL/sect -prepare primary solution: 3% BSA in PBS, 1:50 antibody (thaw as needed)

15 Apply primary antibody O/N @ 4o -1 dip in PBS, blot liquid, add 40uL/sect

DAY 2

16 Dip slides into PBST 10 sec

17 Rinse slides with PBST (x4) 3, 5, 5, 7 -PBS 0.5 stock in cabinet above heater -PBS (in beaker): 700mL water, 200mL PBS, 100 mL NaCl→pH to 7.2-7.6 -Make secondary antibody: 3% BSA, 1.5% NHS, 1:1200 secondary

18 Apply secondary antibody to slides in incubation box 1 hr @ 3¬¬7o -with 15 min left, make ABC: 1% A, 1% B, PBS

19 Dip in PBST (use last wash) 10 sec

20 Rinse slides with PBST (x4) 3, 5, 5, 7 min

21 Apply ABC solution to slides in incubation box 1 hr @ 3¬¬7o

22 Dip in PBST (use last wash) 10 sec

23 Rinse slides with PBST (x4) 3, 5, 5, 7 min -prepare DAB: 2 transfer pipets, 2 cups (waste & DAB), waste ziplock, DAB -DAB in black box in fridge→1 drop/solvent -need 5.7 mL/22 slides -get empty slide container and fill with PBST for stopping DAB rxn

24 Drain and wipe back of slides, place in incubation box, add DAB 2 min

25 Drain excess solution into waste container, use transfer pipet to rinse slides with Tween

26 Rinse slides with PBST (x4) 3, 5, 5, 7 min

27 Put slides in slide holder & dip x5 in 70% EtOH

28 Place in 90% EtOH (x4) 2 min each

29 Place in 100% EtOH (x2) 1 min each

30 Place in 100% EtOH (x2) 3 min each

31 Place in xylene (x5) 5, 2, 2, 2, 2 min

32 Coverslip slides O/N @ RT -2 drops of paramount, careful of bubbles, make sure coverslips don’t slip