CD Marker

Chart hierarchically listing the different types of hematopoietic cells and indicating how to differentiate them. Read on for more details.
(full size chart)

Aim: to determine the distribution and pattern of CD markers in mouse spleen from wild type or test mouse, using enzyme labels for BRIGHTFIELD MICROSCOPY


  1. Frozen sections air dried and used, after 30 minutes or the next day or stored at minus 80 degrees for use in 2 weeks
  2. 10% goat serum, 1%BSA/PBA, blocking buffer
  3. PBS, washing buffer

Positive control and : Negative control: frozen sections of wild type mouse spleen: one slide for no primary antibody and one slide for each CD marker


1. Biotinylated CD3e (Tcells) BDPharmingen Cat. No. 553059 –-use at 2.5 ug/ml --1:200

2. Biotinylated CD4(L3T4/(RM4-5) ; BD Pharmingen Cat. No. 553045 at 1 ug/ml; -1: 500

3. Biotinylated CD8a (Ly-2) ; BD Pharmingen Cat. No. 553029 use at 2.5 ug/ml;-----1:200

4. Biotinylated CD45/B220( B cells RA3-6B2); BDPharmingen Cat. No. 553086 at 2.5ug/ml--1:200

5. Biotinylated CD11b/Mac-1 (monocytes and activated neutrophilsM1/70); BD Pharmingen Cat. No.553309 at 1ug/ml—1:500

6. Biotinylated F480 (resident macrophages); BioSource International Cat. No.AMU 0089 use at--1:50.

And Serotec Cat. No. MCA497B

7. Biotinylated Gr-1(neutrophils/ granulocytes Ly-6G) ; BD Pharmingen Cat. No. 553125 use at----1:200

8. Rabbit anti AsialoGM1 for NK cells; WAKO Cat. No. 986-10001 use at --1:4000

9. Positive control: Biotinylated rat anti mouse CD 31 ) use at ----1:500 BD Pharmingen Cat. No. 09331A (Cat No. 01951D also works well )

10. Negative control: slide receives buffer alone, followed by secondary antibody


  1. Air dry frozen sections for 30 minutes
  2. Fix in acetone (Fisher Cat. No. A16-4) for 10 minutes,
  3. Wash in PBS, 3 changes
  4. Remove endogenous peroxidases by immersing in 0.03% H2O2 for 30 minutes
  5. Wash in PBS, 3 changes
  6. ??????Set up on autostainer
  7. Overlay tissue sections with 1%BSA/ PBS
  8. Remove endogenous biotin by incubating first with 0.1% avidinin PBS for 15 minutes,
  9. followed by 3 PBS washes
  10. then by incubating with 0.01% biotin in PBS for 15 minutes
  11. followed by 3 PBS washes
  12. Overlay with antibodies diluted in 1% BSA/PBS
  13. Incubate for 30 minutes at room temperature
  14. Wash 3 times in PBS
  15. IF any slide received NON-biotinylated anti CD antibodies, the specific binding has to be detected using a Biotinylated Goat anti-Rat 1:500 (BectonDickinsonPharmingen Cat No.554-014)
    FOR WAKO’s anti Asialo GM1, use HRP anti Rabbit secondary at 1: 100
  1. Incubate primary antibodies for 30 minutes at room temperature
  2. Wash 3 times in PBS
  3. Incubate with HRP Streptavidin 1:500 (Jackson Immunoresearch Cat No.016-030-084)
  4. Wash 3 times with PBS
  5. Overlay with VIP for 2-6 minutes (purple substrate--Vector labs Cat No. SK 4600) or the usual AEC substrate--red substrate.
  6. Wash 3 times with PBS
  7. Counterstain nuclei with Mayer’s hematoxylin
  8. Wash 3 times with PBS
  9. Coverslip using Aquamount (Fisher Cat. No BM-01) Or 50% glycerol/PBS)

The positive control tissue section with anti CD31 should demonstrate blood vessels only, as positive staining control. The negative tissue control should only show counter-stained nuclei

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