Immunocytochemistry/ Immunofluorescence abreview for Anti-Stromal interaction molecule 1 antibody

Immunocytochemistry/ Immunofluorescence

Human Cell (B and T lymphocytes)

Yes - Triton / MeOH

B and T lymphocytes

Serum as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 20°C

Formaldehyde

1 hour(s) and 0 minute(s) · Temperature: 20°C · Diluent: 1% BSA in PBS

10 µg/ml

4 µg/ml

Non-Abcam antibody was used: anti-rabbit IgG(H+L)
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor® 555

Information on the image: immune synapse formed between cytotoxic T cell (atop) and B cell target. Green: anti-Perforin conjugated to Alexa488 Ab Red: anti-STIM1 + secondary anti-rabbit Alexa555. My judgement of the specificity of this staining is poor because anti-STIM1 staining does not show ER-like pattern. STIM1 should be associated with ER, and ER staining is very characteristic (large compact vesicular pool).

Thank you for your review of a previously untested application of the antibody, immunocytochemistry/IF. A different fixation protocol, for instance acetone fixation, might give a different result but we have not tried this. It seems likely the antibody in this case stained something other than, or in addition to, STIM1. But STIM1 is not located exclusively in the ER. According to a note in the UniProt record of the human protein, it "translocates from the endoplasmic reticulum to the cell membrane in response to a depletion of intracellular Ca2+ and is detected at punctae corresponding to junctions between the endoplasmic reticulum and the cell membrane." http://www.uniprot.org/uniprot/Q13586