Immunostaining for Hif1 Alpha

September 29, 2020

Vascular Dysfunction
University of Heidelberg
Postdoctoral Researcher

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Company:

R&D

Product Name:

Hif1 alpha

Catalog Number:

AF1935

Clone Number:

N/A

To determine the hypoxia levels by immunostaining with Hif1-alpha in embryonic brain sections

Experimental Design and Results Summary

Applications

Immunofluorescence

Sample

Frozen embryonic mouse brain tissue (E13)

Primary Incubation

1:200, 4 degrees overnight

Blocking Agent

20% Normal rabbit serum in PBST (0.5% Triton X 100)

Secondary Incubation

1:100 anti goat-biotin

Tertiary Incubation

1:100 streptavidin, amplified using perkin elmer amplification reagent

Detection

Immunofluorescence

Results Summary

The sections were treated with antigen retrieval buffers (1M HCL), and then treated with 6% H2O2 in Methanol, and blocked in 20% rabbit serum in mild buffer (TBST) -preferably use blocking and other protocols from Perkin Elmer antibody signal amplification kit. Embryonic brain cortex at early stages shows hypoxia in the progenitor domain which can be distinguished from the other regions of the cortex where blood vessels are present.

DOI or PMID #

N/A

Additional Notes

Multiple antigen retrievals may be necessary to obtain best signals. The amplification of the signal ( using amplification kits) should be optimized by the user

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Summary

The Good

Good for thin cryo sections

The Bad

The signal may not be consistent always, tedious due to multiple antigen retrieval steps

The Bottom Line

The antibody works good when standardized, however may not be used alone as a marker of hypoxia. Other methods like PIMO, or qPCR methods should be combined for hypoxia studies.

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