Immunohistochemistry: CD45

Tissue preparation:

1.  Fix the bone samples in 4% PFA for 24 hrs at 4°C.

  1. Change to fresh PFA and fix for another 24 hrs at 4°C.
  2. Decalcify the samples with 20% EDTA at 4°C for 7 days (change the EDTA daily)

4.  Paraffin processing (histocore)

Before starting:

·  Make 1 L 1X TBS

o  100 ml 10X TBS

o  900 ml dH2O

·  Prepare humid chambers (with Whatman paper and 1X TBS)

·  Make 4 L 1XTNT

o  400 ml 10X TBS

o  3600 ml dH2O

o  2 ml Tween-20 (add while stirring!)

Staining process: (use TSA kit, Perkin Elmer #NEL700A)

1)  Incubate slides at 60°C on slide warmer for at least 1hr.

2)  Deparaffinize and dehydrate (use clean solutions!)

·  Xylene, 3x 3 min

·  100% EtOH, 3x 2 min

3)  Rehydrate in 1X TBS, 5 min RT

·  At this time, prepare PK

4)  Circle sections with Pap-pen

5)  Proteinase K 20ug/ml, 15min RT in moist chamber

·  6 ml of 10mg/ml in 3 ml dH2O

6)  3% H2O2/ MeOH 10min RT

·  25 ml 30% H2O2 + 225 ml MeOH

7)  dH2O, 5min, RT

8)  Equilibrate sections by rinsing with 1X TBS

9)  Incubate with TNB at RT in moist chamber for 30 min.

10) 1° antibody diluted in TNB, RT in moist chamber for 1hr

·  CD45 (BD 550539) diluted 1/200 in TNB

11) Wash: 1X TNT, 3x 5min at RT (on rocking platform)

12) Biotinylated 2° Ab diluted in TNB, in moist chamber for 30 min

·  antiRat IgG,1/300 (Dako, #E0468)

13) Wash: 1X TNT, 3x 5min at RT (on rocking platform)

14) SA-HRP (from kit) diluted in TNB 1/100 30min RT in moist chamber

15) Wash: 1X TNT, 3x 5min at RT (on rocking platform)

16) Biotynil Tyramide (kit), diluted in amplification diluent, 1:50); 5 min RT in moist chamber

17) Wash: 1X TNT, 3x 5min at RT (on rocking platform)

18) SA-HRP (from kit) diluted in TNB 1/100 30min RT in moist chamber

19) Wash: 1X TNT, 3x 5min at RT (on rocking platform)

20) For HRP detection, use DAB (VECTOR kit)

Vector DAB

To 5 ml dH2O:

§  add 2 drops buffer; mix well

§  add 4 drops DAB; mix well

§  add 2 drops H2O2mix well

§  add 2 drops NiCl (optional, if not using hematoxylin counterstain turns DAB color from brown to black)

21) Incubate until color develops (2-10min.)

22) Wash 5 min in dH2O.

23) Counter stain with hematoxylin (Invitrogen 00-8001) for 3 min.

24) Wash 5 min in cold tap water

25) Incubate in 1X PBS 1 min

26) Wash in dH2O

27) Dehydrate (1min in EtOH for 3 times, 2 mins in Xylene for 3 times) and coverslip as usual

Updated 4/21/15 by LM