Mast Cell Tryptase Antibody For Immunofluorescence

Boston Children's Hospital

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Company:

Santa Cruz Biotechnology

Product Name:

Mast Cell Tryptase Antibody (AA1)

Catalog Number:

sc-59587

Identification of mast cells in formalin-fixed paraffin-embedded human tissues is normally accomplished by specialized histological stains (toluidine blue, chloroacetate esterase) that bind to granule components. In this study, I wanted to assess the expression of other proteins in mast cells, and therefore needed an immunofluorescent approach to allow simultaneous analysis of multiple markers. Tryptase is a reliable marker by immunohistochemistry, and can be stained by the AA1 clone.

Experimental Design and Results Summary

Applications

Immunofluorescence

Sample

FFPE human tissue (intestine, skin)

Primary Incubation

Epitope retrieval with citrate buffer, pH 6. Overnight staining at 4C with 1ug/ml primary antibody.

Blocking Agent

2% BSA with 0.1% triton X-100 in PBS.

Secondary Incubation

Biotinylated rat anti-mouse IgG1, clone RMG-1, Biolegend, 2.5ug/ml for 1hr at room temperature.

Tertiary Incubation

Goat anti-rat IgG (H+L) Alexa Fluor 488, ThermoFisher, 10ug/ml for 30min at room temperature.

Detection

Nikon E800 Eclipse microscope

Results Summary

Bright annular staining with granules visible at high magnification. Modest background that could be from the RMG-1 anti-mouse IgG1 secondary (it isn't cross-adsorbed against human). Suitable for use in immunofluorescence. I tested epitope retrieval with citrate buffer pH 6 and EDTA pH 8. Both worked. Epitope retrieval may not be necessary for detection of tryptase, but it will be required for other antigens, and at least the tryptase epitope is robust. The staining also worked on frozen sections.

Additional Notes

None

Related Categories

Image Gallery

Summary

The Good

Very bright

The Bad

Some background in the lamina propria of the intestine that could be improved with optimization.

The Bottom Line

Great antibody. Versatile.

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