Validation Report #029634

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CD4 Molecule (CD4) (ABIN671376) has been independently validated for IHC by the Science Exchange network of experts.

The Science Exchange network is used to independently validate research reagents, protocols, and results to ensure accuracy and reproducibility.

Validation number: #029634
Validated on: 03/26/14

Summary

Antigen

CD4 Molecule (CD4)

Catalog number
Supplier

Bioss

Supplier catalog number
Lot number

130703

Method validated
Laboratory
Validation number
Positive Control
Negative Control

Mouse muscle tissue

Notes

Signal was detected in positive control sample and not in negative control sample.

Independent Results

Full Methods

Primary Antibody

  • Antibody: CD4 Molecule (CD4)
  • Catalog number: ABIN671376
  • Supplier: Bioss
  • Supplier catalog number: bs-0647R
  • Lot number: 130703

Isotype Control Antibody

  • Antibody: Rabbit IgG isotype control
  • Supplier: Bioss
  • Catalog number: bs-0295P
  • Lot number: YYDW72P

Secondary Antibody

  • Antibody: Donkey anti-Rabbit IgG Antibody (Biotinylated)
  • Supplier: Jackson ImmunoResearch
  • Catalog number: 711-065-152

Controls

  • Positive control: Spleen tissue (specimen known to contain the target protein) from mouse.
  • Negative Control: Muslce tissue (specimen known to not contain the target protein) from mouse.
  • Primary antibody isotype control: Mouse spleen treated with primary antibody isotype control instead of the primary antibody.
  • Secondary antibody only control: Mouse spleen treated with secondary antibody only (no primary antibody).

Protocol

  • Immunohistochemistry was performed manually.
  • Sections were deparaffinized and rehydrated.
  • Sections were heated to 98°C for 10 min in 0.1M Borate buffer with 1 mM NaCl and 1 mM EDTA pH 8.0 for antigen retrieval.
  • Sections were incubated in 3% hydrogen peroxide for 10 min at room temperature to block endogenous peroxidases.
  • Sections were washed x 3 in Tris buffered saline (TBS).
  • Sections were blocked in 5 % normal donkey serum for 60 min at room temperature.
  • Sections were incubated with primary antibody diluted 1:200 in 5% normal donkey serum in TBS. Incubated at 4°C temperature overnight.
  • Sections were washed x 3 in Tris buffered saline.
  • Sections were incubated with secondary antibody diluted 1:400 in 5% normal donkey serum in TBS. Incubated for 60 min at room temperature..
  • Sections were washed x 3 in Tris buffered saline.
  • Sections were incubated with Vectastain ABC kit (Vector Lab PK6100) to enhance signal.
  • Sections were incubated with DAB chromogenic substrate (Vector Lab SK-4105) for 60 sec at RT.
  • Sections were washed x 3 in Distilled Water.
  • Sections were counterstained with hematoxylin for 20 sec.
  • Sections were washed x 1 in Distilled Water.
  • Sections were dehydrated, mounted and photographed under a Zeiss AxioImager M1 light microscope.

Experimental Notes

  • No experimental challenges noted.

Figures

1395768622675 dae9f98864de82a2b2302378caa1eb0f.tif?fit=fill&mark=%2freproducibility%2ftsen logo
Figure 1: Mouse spleen stained with anti-CD4 (brown) and counterstained with hematoxylin.
1395768622687 91635056747edc7079f0c099504a8d0b.tif?fit=fill&mark=%2freproducibility%2ftsen logo
Figure 2: Mouse muscle stained with anti-CD4 (brown) and counterstained with hematoxylin.
1395768622690 3e37d768b97a05ef97be27e3d723f0e0.tif?fit=fill&mark=%2freproducibility%2ftsen logo
Figure 3: Mouse spleen stained with isotype control antibody (brown) and counterstained with hematoxylin.
1395768622692 4ba539c35c7034959f5b63661d5baaf6.tif?fit=fill&mark=%2freproducibility%2ftsen logo
Figure 4: Mouse spleen stained with secondary antibody only (brown) and counterstained with hematoxylin.
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