Reliable GFP Antibody for Brain Tissue (Immunostaining)

February 21, 2025

BlueskyReddit
Physiology and Biophysics
State University of New York at Buffalo
Postdoctoral Researcher

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Company:

ThermoFisher Scientific / Invitrogen

Product Name:

GFP Recombinant Rabbit Antibody

Catalog Number:

G10362

Clone Number:

Monoclonal Antibody

The GFP antibody was used for immunostaining of mouse brain astrocytes to visualize which astrocytes were transduced with AAV5-GFAP(0.7)-eGFP (virus injected into the brain region of interest - prefrontal cortex). Immunostaining was part of the BaseScope Red Assay combined with Immunohistochemistry (Integrated Co-Detection Workflow). The BaseScope assay workflow utilizes a peroxide step that degrades any endogenous reporter fluorescence, thus requiring additional GFP immunostaining. The GFP-expressing virus was under the GFAP promoter (astrocyte-specific), thus only astrocytes were GFP-positive.

Experimental Design and Results Summary

Applications

Immunofluorescence

Sample

Mouse brain slices (15 µm thickness), fresh frozen.

Primary Incubation

Dilution 1:20; incubation overnight at 4°C.

Blocking Agent

The antibody was diluted in Antibody Diluent (ACD; Bio-Techne, REF: 323160, part of the kit).

Secondary Incubation

Alexa 488 diluted 1:1000 in Antibody Diluent (ACD; Bio-Techne, REF: 323160, part of the kit).

Tertiary Incubation

N/A

Detection

Images were obtained on an inverted confocal microscope Leica-Sp8 using LASX software.

Results Summary

This GFP antibody allowed us to identify which astrocytes were transduced with the AAV5-GFAP(0.7)-eGFP virus into the mouse brain. Using this antibody, we were able to integrate immunostaining into our BaseScope protocol, which allowed us to quantify mRNA of interest in a cell-type-specific manner (GFP+ astrocytes).

DOI or PMID #

N/A

Additional Notes

N/A

Image Gallery

Summary

The Good

Works reliably and provides good immunostaining.

The Bad

Requires high concentrations to work in the integrated BaseScope workflow.

The Bottom Line

The antibody is quite expensive considering the low dilution used (high concentration), but provides the expected immunostaining and is not sensitive to the BaseScope harsh reagents, allowing integration of immunostaining into the workflow.

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