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

Material:

  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

Antibodies:

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

Procedure:

  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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