The influence of protease digestion and duration of fixation on the immunostaining of keratins. A comparison of formalin and ethanol fixation.

Author(s): Battifora H, Kopinski M

Publication: J Histochem Cytochem, 1986, Vol. 34, Page 1095-100

PubMed ID: 2426335 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of protease digestion as well as the duration and type of fixation on immunohistochemical (IHC) staining of keratins. Postmortem tissue brain, kidney, esophagus, stomach, pancreas, lung, prostate, muscle, and skin from an adenocarcinoma patient and an infiltrating ductal carcinoma of the breast were examined.

   Summary of Findings:

   For ethanol-fixed tissues, strong keratin immunostaining and structural details were observed regardless of the duration of fixation. However, protease digestion led to disintegration of cell structure and inhibited immunostaining regardless of protease used and duration of digestion. For formalin-fixed tissues, weak or no keratin immunostaining was observed without protease digestion. All three proteases enhanced immunostaining of formalin-fixed tissues. The required digestion time, in order to achieve optimum staining, increased with longer fixation duration. Digestion of tissues with pepsin required less time than when trypsin or pronase were used. Longer incubations with trypsin resulted in increased background staining and overdigestion with any of the three proteases led to loss of cytological details. The exact digestion time required for any fixation duration was dependent on the tissue examined.

   Biospecimens

   • Tissue - Brain
   • Tissue - Esophagus
   • Tissue - Stomach
   • Tissue - Breast
   • Tissue - Lung
   • Tissue - Prostate
   • Tissue - Muscle (Skeletal)
   • Tissue - Skin
   • Tissue - Kidney
   • Tissue - Pancreas

   Preservative Types

   • Ethanol
   • Formalin

   Diagnoses:

   • Autopsy
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Ethanol Formalin (buffered)
   Biospecimen Preservation	Time in fixative	3 h 8 h 9 h 12 h 24 h 1 week 3 weeks 6 weeks
   Analyte Extraction and Purification	Protein digestion	None Trypsin Pepsin Pronase 5-180 min
   Biospecimen Acquisition	Biospecimen location	Brain Kidney Esophagus Stomach Pancreas Lung Prostate Muscle Skin Breast

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