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Updated: Aug 8, 2026
Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections
Published on: November 14, 2016
Quantitative comparison of immunohistochemical staining intensity in tissues fixed in formalin and Histochoice
D G Vince1, A Tbakhi, A Gaddipati
1Whitaker Biomedical Imaging Laboratory, Department of Biomedical Engineering, Cleveland Clinic Foundation, Ohio 44195, USA. vince@bme.ri.ccf.org
Insights
Histochoice tissue fixative demonstrates comparable or superior staining intensity compared to neutral buffered formalin (NBF). This study quantifies Histochoice
Area of Science:
- Histology and Immunohistochemistry
- Tissue Fixation Techniques
- Biomolecular Analysis
Background:
- Formaldehyde-based fixatives like neutral buffered formalin (NBF) are standard for tissue preservation.
- Formaldehyde cross-linking can reduce tissue antigenicity and immunohistochemical stain intensity.
- Histochoice is a potential alternative fixative suggested to enhance staining.
Purpose of the Study:
- To quantitatively compare the immunohistochemical staining intensity of Histochoice and NBF.
- To statistically evaluate differences in hue, saturation, and value between the two fixatives.
- To determine if Histochoice offers superior staining compared to NBF.
Main Methods:
- Tissue samples were bisected and fixed using either NBF or Histochoice.
- Sections were stained with 21 different antibodies.
- Color images were analyzed to determine hue, saturation, and value of positive pixels.
- Analysis of Variance (ANOVA) was performed for statistical comparison.
Main Results:
- Histochoice showed greater mean saturation than NBF for 57.1% of antibodies tested.
- No significant saturation difference was observed in 28.6% of cases.
- Histochoice resulted in lower values (darker staining) in 66.7% of cases compared to NBF.
- Minor hue differences were noted in 8 out of 21 antibodies.
Conclusions:
- Histochoice provides immunohistochemical staining intensity comparable to NBF.
- Histochoice fixation may offer superior staining intensity in a significant number of cases.
- This fixative is a viable alternative for preserving tissue while maintaining or enhancing stain quality.
Abstract:
Formaldehyde fixatives have traditionally been used to preserve tissues as they impart excellent morphological preservation. Formaldehyde fixes tissue by cross linking, a process which can reduce the antigenicity of tissue and weakens the intensity of immunohistochemical stains. Preliminary studies have shown that Histochoice tissue fixative offers equal or greater staining intensity than neutral buffered formalin (NBF). This study compares these fixatives quantitatively and presents the results in unambiguous statistical terms. Tissue samples were collected, bisected, and fixed in NBF or Histochoice. The sections were stained with a total of 21 antibodies, and color images were collected. The hue, saturation, and value were determined for each positive pixel and an ANOVA performed. Small differences in hue were noted in 8 of 21 cases. Histochoice-fixed tissue gave a greater mean saturation than NBF with 57.1% of the antibodies tested. No significant difference in the saturation was detected in 28.6% of the cases; NBF gave higher mean saturation levels with only 14.3% of the antibodies. Histochoice-fixed tissue was found to give lower values in 66.7% of cases than those prepared with NBF, indicating darker staining. These results show that Histochoice produces staining intensity that is comparable, and in many cases superior, to formalin.
