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Validated IHC

LSBio have created 2 ranges of IHC validated antibodies for FFPE and shared their method for easy replication. 

LSBio have taken the sting out of finding good quality antibodies for your research with their validated ranges of antibodies. With over 20,000 These antibodies are tested on over 20 normal human tissue types and more, as specific to the antibody. To be selected for the IHC-plus™ range the antibody must have a close correlation to the published literature, be high affinity, show minimal staining artifacts and have a high signal to noise ratio.

The Path Plus™ Range offers the highest quality and best in class staining, as well as fulfilling all the criteria for the IHC-plus range.  

2BScientific offer in house IHC support. They’re always happy to discuss your method to help it meet your needs.

Tissue Preparation

  • Formalin fixation and embedding in paraffin wax

Tissue Sectioning

  • Make 4-µm sections and place on pre-cleaned and charged microscope slides
  • Heat in a tissue-drying oven for 45 minutes at 60°C

Deparaffinization & Rehydration

  • Wash slides in 3 changes of xylene* – 5 minutes each at room temperature
  • Wash slides in 3 changes of 100% alcohol – 3 minutes each at room temperature
  • Wash slides in 2 changes of 95% alcohol – 3 minutes each at room temperature
  • Wash slides in 1 change of 80% alcohol – 3 minutes at room temperature
  • Rinse slides in gentle running distilled water – 5 minutes at room temperature

Antigen Retrieval

  • Steam slides in 0.01 M sodium citrate buffer, pH 6.0 at 99-100°C - 20 minutes
  • Remove from heat and let stand at room temperature in buffer - 20 minutes
  • Rinse in 1X TBS with Tween (TBST) – 1 minute at room temperature

Immunostaining

  • Do not allow tissues to dry at any time during the staining procedure
  • Apply a universal protein block – 20 minutes at room temperature. 
  • Drain protein block from slides, apply diluted primary antibody – 45 minutes at room temperature.
  • Rinse slides in 1X TBST - 1 minute at room temperature.
  • Apply a biotinylated secondary antibody (specific to the host of the primary antibody) - 30 minutes at room temperature
  • Rinse slides 1X TBST – 1 minute at room temperature.
  • Apply alkaline phosphatase streptavidin (AK-5000) – 30 minutes at room temperature
  • Rinse slides in 1X TBST - 1 minute at room temperature
  • Apply alkaline phosphatase chromogen substrate (SK-5100) – 30 minutes at room temperature
  • Wash slides in distilled water – 1 minute at room temperature

Dehydrate

  • This method should only be used if the chromogen substrate is alcohol insoluble.
  • Wash slides in 2 changes of 80% alcohol – 1 minute each at room temperature.
  • Wash slides in 2 changes of 95% alcohol – 1 minute each at room temperature.
  • Wash slides in 3 changes of 100% alcohol – 1 minute each at room temperature.
  • Wash slides in 3 changes of xylene – 1 minute each at room temperature.
  • Apply ‘permanent’ / non aqueous mounting media.
  • Apply coverslip

 

LSBio and Vector Laboratories

LSBio offers Vector Laboratories reagents in the USA, to enable researchers to source antibodies & detection from one place. 2BScientific is happy to be able to exclusively supply the Vector Laboratories' reagents in the UK.

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