Porcine intestinal mast cells. Evaluation of different fixatives for histochemical staining techniques considering
J Rieger1, S Twardziok, H Huenigen
1Freie Universität Berlin Institute for Veterinary Anatomy Koserstr. 20 D-14195 Berlin, Germany. Juliane.Rieger@fu-berlin.de.
Abstract:
Staining of mast cells (MCs), including porcine ones, is critically dependent upon the fixation and staining technique. In the pig, mucosal and submucosal MCs do not stain or stain only faintly after formalin fixation. Some fixation methods are particularly recommended for MC staining, for example the fixation with Carnoy or lead salts. Zinc salt fixation (ZSF) has been reported to work excellently for the preservation of fixation-sensitive antigens. The aim of this study was to establish a reliable histological method for counting of MCs in the porcine intestinum. For this purpose, different tissue fixation and staining methods that also allow potential subsequent immunohistochemical investigations were evaluated in the porcine mucosa, as well as submucosa of small and large intestine. Tissues were fixed in Carnoy, lead acetate, lead nitrate, Zamboni and ZSF and stained subsequently with either polychromatic methylene blue, alcian blue or toluidine blue. For the first time our study reveals that ZSF, a heavy metal fixative, preserves metachromatic staining of porcine MCs. Zamboni fixation was not suitable for histochemical visualization of MCs in the pig intestine. All other tested fixatives were suitable. Alcian blue and toluidine blue co-stained intestinal goblet cells which made a prima facie identification of MCs difficult. The polychromatic methylene blue proved to be the optimal staining. In order to compare MC counting results of the different fixation methods, tissue shrinkage was taken into account. As even the same fixation caused shrinkage-differences between tissue from small and large intestine, different factors for each single fixation and intestinal localization had to be calculated. Tissue shrinkage varied between 19% and 57%, the highest tissue shrinkage was found after fixation with ZSF in the large intestine, the lowest one in the small intestine after lead acetate fixation. Our study emphasizes that MC counting results from data using different fixation techniques can only be compared if the respective study-immanent shrinkage factor has been determined and quantification results are adjusted accordingly.
Insights
Zinc salt fixation (ZSF) effectively stains porcine mast cells (MCs) in the intestine. Polychromatic methylene blue is the optimal stain, but tissue shrinkage must be accounted for when comparing results.
Area of Science:
- Histology
- Veterinary Pathology
- Immunohistochemistry
Background:
- Mast cell (MC) staining in porcine tissues is challenging with standard formalin fixation.
- Previous studies suggest Carnoy or lead salt fixation for MCs.
- Zinc salt fixation (ZSF) shows promise for preserving sensitive antigens.
Purpose of the Study:
- To establish a reliable histological method for counting porcine intestinal mast cells (MCs).
- To evaluate various fixation and staining techniques for MC visualization and potential immunohistochemistry.
- To compare the efficacy of different fixatives and stains in porcine mucosa and submucosa.
Main Methods:
- Porcine intestinal tissues were fixed using Carnoy, lead acetate, lead nitrate, Zamboni, and ZSF.
- Tissues were stained with polychromatic methylene blue, alcian blue, or toluidine blue.
- Tissue shrinkage was measured and shrinkage factors calculated for each fixation and intestinal location.
Main Results:
- Zinc salt fixation (ZSF) enabled metachromatic staining of porcine mast cells (MCs) for the first time.
- Polychromatic methylene blue was the optimal stain; alcian blue and toluidine blue stained goblet cells.
- Zamboni fixation was unsuitable; Carnoy, lead salts, and ZSF were suitable.
- Tissue shrinkage varied significantly (19-57%), necessitating specific correction factors.
Conclusions:
- Zinc salt fixation (ZSF) is a reliable method for preserving and visualizing porcine intestinal mast cells (MCs).
- Polychromatic methylene blue is the recommended stain for mast cell enumeration.
- Accurate comparison of mast cell counts across different fixation methods requires accounting for study-immanent tissue shrinkage.
