Methods

Standard Operating Procedures (SOPs):

  • Guidelines for the Submission to the Koch Histology Lab (PDF) (Word)
  • Filling out the iLab forms (PDF) (Word)
  • Types of tissue cassettes and how to use them (PDF) (Word)
  • Sample drop-off checklist (PDF) (Word)
  • Suggested sectioning for Tissues for Submission to the Koch Histology Lab (PDF) (Word)
  • IHC Checklist v7  (PDF) (Word)
  • Suggested Guidelines for Processing Tissues to Freezing for Future Cyrosections (PDF) (Word)
  • Histogel processing of Histology and Cytology specimens (PDF) (Word)
  • Quickstart guide for accessing whole slide images (PDF) (Word)
  • Using the Sakura stainer and coverslipper (PDF) (Word) (First-time users, contact the Histology Facility to obtain training and authorization.)

 

Paraffin-Embedded Tissues

This medium is appropriate for most routine histology. Fixed tissue pieces are dehydrated through a series of graded alcohols before clearing with xylenes. The final step in the processing is the replacement of the xylenes with molten paraffin. The tissue is then removed from the processing machine, infiltrated with fresh molten paraffin, oriented, and allowed to cool and harden into a block. Tissue is then sectioned on a microtome and slides are produced.

 

How to Section using a Microtome:

The Hope Babette Tang Histology Core Presents: How to Section using a Microtome - a brief overview on how to section paraffin-tissue embedded blocks using a microtome.


Frozen Sections

Many antigens and RNA do not survive routine histological processes. An alternative method requires tissue to be frozen and sectioned in a cryostat. Antigen preservation is always superior in fresh frozen tissue. Unfortunately, the drawback to frozen sections is the vastly inferior morphology of the tissue.

 

What you need to do:

 

Tissue can be dissected, placed in a disposable mold that has been labeled as previously discussed, immersed in OCT (a freezing medium), and flash frozen in a bath of dry ice and liquid nitrogen. Once again, orientation must be considered before freezing the tissue. Variations to this protocol include fixation in 10% formalin followed by immersion in various concentrations of sucrose and slow freezing on dry ice. We can help you determine the best method to demonstrate the particular antigen in a specific tissue.

Tissue should be stored at -80°C until it is sectioned.

 

How to Section in the Cryostat:

The Hope Babette Tang Histology Core Presents: How to Section in the Cryostat - a brief overview on how to section frozen specimens. 


Resin-embedded sections

The facility has available a Thermo automated microtome and tungsten carbide blade used in sectioning resin-embedded tissue for the light microscope. The reagents used are glycol methacrylate resins that produce a firmer medium, allowing thinner (2 micron) sections to be cut. It is particularly useful for undecalcified bone. The disadvantages of this method involve the toxicity of the reagents and the limited stains that are compatible with the resin.

 

What you need to do:

 

At this time, we do not process or embed tissue in resin, but we can section this material after you process and embed it. We can help you decide on appropriate stains for use with resin-embedded tissue.


AnchorPathology Consultation

The Koch Institute has an affiliation with Dr. Roderick Bronson, a veterinary pathologist at Harvard Medical School. He is available to consult with investigators on most Fridays. Appointments are requested through iLab (Schedule Resources tab).

We hope this brief overview will help get you started with histology. If you have other concerns or issues you would like to discuss or if you would to observe the histology operation, please feel free to contact us at X8-8183.