Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Evaluation of tissue preparation methods and paired immunofluorescence staining for immunocytochemistry of lymphomas.

Author(s): Brandtzaeg P, Rognum TO

Publication: Histochem J, 1983, Vol. 15, Page 655-89

PubMed ID: 6350234 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 fixative type on IF staining in normal colon mucosa, tonsils, nasal mucosa, and lymphoma specimens. Tissues were fixed in ethanol or in Baker's fluid at 4 degrees C while all other fixation occurred at room temperature. The duration and concentrations of ethanol used for dehydration varied based on fixative type. Sections of ethanol-fixed specimens were dried for a shorter amount of time than sections of specimens fixed in other fixatives.

   Summary of Findings:

   IF staining of IgG, IgA, IgM, and alpha-naphthylbutyrate esterase was best in ethanol-fixed specimens and was improved by a 48 h PBS wash prior to fixation. Fixation with formalin, formalin-sublimate, Bouin's, and acetic acid-formalin-saline required an 8-fold higher concentration of conjugates to mimic staining observed in the prewashed, ethanol-fixed specimens. J-chain and epithelial secretory component staining were best in ethanol- or carbodiimide-fixed specimens, but J-chain staining needed pretreatment with acid urea. Fixation with Baker's formalin-calcium, carbodiimide, or acetic acid-formalin-saline yielded the best IgD immunostaining. Fixation with all fixatives except gluteraldehyde resulted in good light chain immunostaining. For most cross-linking fixatives, increasing conjugate incubation time from 30 min to 20 h (at the same concentration) improved signal to noise ratios. In formalin-fixed tissues, pronase treatment was able to improve IgG, IgA, and IgM, but not IgD immunostaining, and J-chain staining was more improved by trypsin digestion than pronase treatment. In tissues fixed with other fixatives, pronase treatment had negative effects on immunostaining. IgM immunostaining was slightly better than IgG and IgA immunostaining for all fixatives.

   Biospecimens

   • Tissue - Colorectal
   • Tissue - Lymph Node
   • Tissue - Head and Neck
   • Tissue - Tonsil

   Preservative Types

   • Ethanol
   • Other Preservative
   • Formalin

   Diagnoses:

   • Neoplastic - Lymphoma
   • Normal
   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Tonsillitis
   Analyte Extraction and Purification	Protein digestion	Pronase treatment Trypsin digestion None
   Biospecimen Preservation	Type of fixation/preservation	Ethanol Formalin (buffered) Gluteraldehyde-formaldehyde Baker's formalin-calcium Formol sublimate Acetic acid-formalin-saline Bouin's fixative Susa fixative Carbodiimide
   Biospecimen Preservation	Temperature of fixation/preservation	Room temperature 4 degrees C
   Biospecimen Aliquots and Components	Tissue section adhesion	Slide drying for 30 min at 37 degrees C Slide drying for 30 min at 56 degrees C and overnight at 37 degrees C
   Immunohistochemistry Specific	Detection method	Standard conjugate concentration 8-fold higher dilution 12-fold higher dilution
   Immunohistochemistry Specific	Incubation time	30 min 20 h
   Analyte Extraction and Purification	Antigen retrieval	Acid urea None
   Immunohistochemistry Specific	Targeted peptide/protein	IgG IgA IgM IgD J chain Kappa light chain Lambda light chain Secretory component Alpha-naphthylbutyrate esterase
   Biospecimen Acquisition	Pre-preservation condition	No wash 48 h PBS wash

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