Validation Report

#029732

Tumor Necrosis Factor (TNF) (ABIN677318) has been independently validated for Immunofluorescence 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: #029732

Validated on: 06/11/14

Summary

Antigen

Tumor Necrosis Factor (TNF)

Catalog number

Supplier

Bioss

Supplier catalog number

Lot number

140113

Method validated

Laboratory

Validation number

Positive Control

Negative Control

Notes

Fluorescent signal is detected in positive control, but not in negative control or isotype controls.

Independent Results

Full Methods

Primary Antibody
  • Antibody: Tumor Necrosis Factor (TNF)
  • Catalog number: ABIN677318
  • Supplier: Bioss
  • Supplier catalog number: bs-2081R
  • Lot number: 140113
Isotype Control Antibody
  • Antibody: Rabbit IgG Isotype
  • Supplier: Bioss
  • Supplier catalog number: bs-0295p
  • Lot number: not available
Secondary Antibody
  • Antibody: Donkey anti-Rabbit IgG (Heavy & Light Chain) Antibody (Alexa 647)
  • Catalog number: 711-605-152
  • Supplier: Jackson Immuno Research
  • Lot number: Not available
Controls
  • Positive control: FFPE normal mouse lung tissue
  • Negative Control: FFPE normal mouse fat tissue
  • Isotype antibody control: tissue sections treated with Rabbit IgG Isotype at 1ug/ul
  • Secondary antibody only control: positive and negative control tissue sections treated with Goat anti-Rabbit Alexa 647 secondary antibody only. Any staining observed is due to non-specific binding of secondary antibody.
Protocol
  • Paraffin embedded positive and negative control tissue sections were deparaffinized and underwent antigen retrieval using 10 mM sodium citrate pH 6.0 in a pressure cooker for 10 min.
  • The tissue sections were incubated with 1 mg/mL sodium borohydride for 10 min at room temperature to block autofluorescent background signal. The sections were then rinsed three times in TBS for 5 min each at RT.
  • Tissue sections were blocked in 1 X TBS / 5% normal donkey serum for 60 min at RT.
  • Tissue sections were incubated with primary antibody diluted 1:200 in 1X PBS / 5% normal donkey serum overnight at 4°C.
  • Tissue sections were rinsed three times in TBS for 5 min each at RT.
  • Cells were incubated with secondary antibody diluted 1:200 in 1X PBS / 5% normal donkey serum for 60 min at RT in dark.
  • Tissue sections were rinsed three times in TBS for 5 min each at RT.
  • Coverslips were mounted on slides with Prolong Gold anti-fade mounting media (Invitrogen).
  • IF stained tissue sections were imaged with a Zeiss LSM 510 Meta confocal microscope.
Experimental Notes

Nothing to note.

Figures

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Panel 1: confocal image of positive control (mouse lung) with anti-TNF alpha antibody

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Panel 2: confocal image of negative control (mouse adipose) with anti-TNF alpha antibody

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Panel 3: confocal image of positive control (mouse lung) with isotype control

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Panel 4: confocal image of negative control (mouse adipose) with isotype control

1402605891119 cfe2403388d63677f6b39aa08c9756a3.tif?fit=fill&mark=%2freproducibility%2ftsen logo

Panel 5: confocal image of positive control (mouse lung) with secondary antibody only