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| Immunohistochemistry Forum Immunohistochemistry Forum. Post and discuss questions on immunohistochemistry methods including paraffin, frozen, or free floating tissue sections. |
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| Hey, I am a first year Phd student and I am trying to perform immunohistochemical staining on tumor tissue harvested from mouse injected with B16F10 melanoma cells.My protocol is as follows : 1. Harvest tissue and embed in OCT medium and freeze in -80C 2. Section of 10 u was taken on + coated slide 3. Section fixed in -20 cooled acetone for 5 min at RT 4. wash 3 times in PBS (5 min each) 5. Blocking with 10% horse serum at RT for 1h 6. Washed 2 times with PBS (5 min each) 7. Add primary Ab (Abcam Rabbit polyclonal to FoxP3 and Biotin Rat antimouse CD4 - both I have tried with 1:300 and 1:500 dilution) for 30 min at RT Ab diluent is PBS + 1%BSA 8. 4 wash with PBS (10 min each) 9. Add secondary Ab (Streptavidin -FITC and DyLight 549 Donkey anti rabbit IgG- I have tried dilution of 1:3000 and 1:8000) Ab diluent is PBS + 1%BSA Ffor 30 min at RT. 10 4 wash with PBS (10 min each) 11 Add DAPI (1:4000) FOR 5 MIN at RT in dark 12 wash with PBS FOR 5 MIN AT rt 13 Incubate with 1%PFA in PBS for 5 min 14 mount in mounting media The problem is that there is there is high bacground and nonspecific staining . Almost everything seem to stain and even where there is no nuclear stain there is still red (foxp3)and green (cd4) staining I am absolutely confused what to do ... please help me with suggestion Thanks in advance |
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| background , cd4 , foxp3 , high , melanoma , mouse , problem , staining , tissue , tumor |
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