Pretreatment for immunohistochemical staining simplified.

Author(s): Boenisch T

Publication: Appl Immunohistochem Mol Morphol, 2007, Vol. 15, Page 208-12

PubMed ID: 17525636 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 deparaffinization, rehydration, and antigen retrieval methods, as well as slide type and section thickness on IHC staining of 23 tissue types using a panel of 18 different antibodies.

   Summary of Findings:

   The use of DakoCytomation Target Retrieval Solution for heat-induced antigen retrieval without prior use of Histo-Clear for deparaffinization resulted in incomplete removal of paraffin. When Histo-Clear was used for deparaffinization followed by heat-induced antigen retrieval with DakoCytomation Target Retrieval Solution (method 1), the silanized slides worked best. Superfrost/Plus and poly-L-lysine-coated slides occasionally caused folding of the tissue sections and showed some retention of paraffin which was more common for tightly organized tissue types and in 5 uM as opposed to 3-4 uM thick sections. Pretreatement method 1 resulted in similar immunostaining intensity to method 5. Despite the recommendation of enzymatic antigen retrieval for 6 antibodies, 5 showed equal or better immunostaining with method 1, which did not include proteinase K digestion, than with method 5 including proteinase K digestion. The exception, antikeratin, showed significantly improved immunostaining after a proteinase K step was included with pretreatment method 1. None of the solutions promoted as accomplishing deparaffinization, rehydration, and antigen retrieval simultaneously, including Trilogy (Cell Marque Corporation), Declere (Cell Marque Corporation), BORGDecloaker (Biocare Medical), 10X Reveal (Biocare Medical), HOT RINSE (Biocare Medical), Pretreatment module deparaffinization and heat-induced epitope retrieval Solution (PS), pH 6.0, pH 8.0, and pH 10.0 (Lab Vision Corporation), worked consistently and all resulted in retained paraffin, folded sections, and even loss of part or all of some sections, regardless of slide type.

   Biospecimens

   • Tissue - Adrenal Gland
   • Tissue - Tonsil
   • Tissue - Brain
   • Tissue - Breast
   • Tissue - Cervix
   • Tissue - Colorectal
   • Tissue - Esophagus
   • Tissue - Heart
   • Tissue - Kidney
   • Tissue - Uterus
   • Tissue - Liver
   • Tissue - Lung
   • Tissue - Ovary
   • Tissue - Pancreas
   • Tissue - Pituitary Gland
   • Tissue - Small Bowel
   • Tissue - Spleen
   • Tissue - Stomach
   • Tissue - Testis
   • Tissue - Thyroid Gland
   • Tissue - Bone Marrow

   Preservative Types

   • Formalin

   Diagnoses:

   • Not specified
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry
   Morphology	Light microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Deparaffinization	Histo-Clear Histo-Clear followed by ethanol Xylene None Prior to antigen retrieval After antigen retrieval Trilogy Declere BORGDecloaker 10X Reveal HOT RINSE PS, pH 6.0 PS, pH 8.0 PS, pH 10.0
   Analyte Extraction and Purification	Antigen retrieval	DakoCytomation Target Retrieval Solution, 97 degrees C Citrate buffer, 97 degrees C
   Analyte Extraction and Purification	Protein digestion	Proteinase K None
   Biospecimen Aliquots and Components	Type of slide	Superfrost/Plus Poly-L-lysine-coated Silanized
   Biospecimen Aliquots and Components	Aliquot size/volume	3 uM section 4 uM section 5 uM section

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