Immunohistochemistry Protocol (Paraffin Sections)

This article shows you how to do IHC (Immunohistochemistry Protocol) for paraffin sections

Immunohistochemistry Protocol (Paraffin)

This article contains IHC protocol for Paffain sections. immunohistochemistry protocol (paraffin sections).

Fixation and Sectioning

  1. Fix dissected tissues in 2% paraformaldehyde, Bouin’s solution, or other fixative for 30 minutes to overnight. (NOTE: Fixation time will depend on species, tissue origin and size of tissue sections).
  2. Embed tissue in paraffin.
  3. Section the tissues 5-10 micrometers thick.
  4. If using paraformaldehyde as fixative antigen retrieval may be necessary.

Deparaffinization and Tissue Rehydration

  1. Deparaffin samples by incubating sections 2-3 times in xylene for 10 minutes each.
  2. Hydrate samples by placing in 100% ethanol for 2 x 3 minutes each, then in 95%, 70%, 50%, 30% ethanol for 1 x 2 minutes each.

Blocking and Primary Antibody Incubation

  1. Block slides with 10% serum from the species from which the secondary antibody was taken or 8% BSA.
  2. Incubate for 30 minutes to 1 hour at room temperature in a humidified chamber.
  3. Wash samples in PBS containing 2% BSA for 5 minutes.
  4. Incubate slides in a humidified chamber overnight with primary antibody solution (primary antibody in PBS containing 2% BSA).
  5. Wash three times with PBS containing 2% BSA for 5 minutes.

Secondary Antibody Incubation and Detection

  1. Incubate slides in a humidified chamber with secondary antibody solution (secondary antibody in PBS containing 2% BSA) according to manufacturer’s protocols.
  2. Wash three times with PBS containing 2% BSA for 5 minutes.
  3. Detect according to manufacturer’s protocols.

 

http://blog.immunoreagents.com/immunohistochemistry-potocol-paraffin/

 

ImmunoReagents, Inc. is a leading global manufacturer of quality antibodies and reagents used in pharmaceutical research, life science research and in vitro diagnostics. Product offerings include a wide range of immunochemistry reagents such as purified immunoglobulinsprimary antibodies, and secondary antibodies covering a broad spectrum of immunoglobulins from various species. ImmunoReagents also provides custom manufacturing to meet specific customer requirements while adhering to cGMP guidelines and ISO quality systems requirements. The company is located in Raleigh, North Carolina