Formalin Versus Bouin Solution for Testis Biopsies: Which Is the Better Fixative?

Abstract

Purpose: Some resources recommended Bouin solution for the fixation of testis biopsy specimens. We compared the histologic quality of rat testicular tissue using buffered formalin and Bouin solution as fixatives. Methods: We prospectively compared the histologic quality of rat testicular tissue fixed in Bouin solution versus formalin. Testicular tissue was harvested post-mortem from six rats. Each testis was removed and sectioned in half; one half was fixed in formalin and one half in Bouin solution. Testicular tissue histology (nuclear membrane detail, nuclear granularity, cytoplasmic granularity, cytoplasmic membrane detail, and basement membrane detail) was graded as high quality (2) or low quality (1). Sloughing of cells into the lumens of the seminiferous tubules was graded on a 0-3 scale (0=none, 1=mild, 2=moderate, 3=extensive). Results: All slides regardless of fixative were of appropriate quality for the histologic evaluation of spermatogenesis. The average sloughing score for formalin cases was 1.4 and for Bouin cases 1.6. Formalin fixed tissue was found to have high quality nuclear membrane detail (2), nuclear granularity (1.9), and basement membrane detail (2). Cytoplasmic granularity was of lesser but adequate quality (1.4). Cytoplasmic membrane detail was poor, (1). Tissue fixed with Bouin solution had high quality basement membrane detail (2) and adequate cytoplasmic granularity (1.5), nuclear membrane detail (1.3) and nuclear granularity (1.4). Cytoplasmic membrane detail was poor (1). Conclusion: Compared to Bouin solution, formalin fixation of rat testicular tissue produced adequate histology for the evaluation of spermatogenesis and may be superior to Bouin solution for certain cytologic features.

The murine male reproductive organ at a glance: Three‐dimensional insights and virtual histology using label‐free light sheet microcopy

“…Although PFA did not change the autofluorescence pattern of, for example, Leydig cells or cell membranes, the nuclear and cytoplasmic granularity increased after PFA fixation compared to results obtained with Davidson's fixative of different concentration. Similar effects were observed by others when using a standard formalin fixation compared to Bouin's solution as fixative, 38 which together with our results demonstrates that careful tissue preparation especially the fixative is an important prerequisite for high quality datasets of the testis.…”

Immunofluorescence Staining of Paraffin Sections Step by Step

“…We achieved the best staining result of the testicular seminiferous tubules using this fixative and after puncturing the thick capsule of the testes ( Figure 5C ; Zaqout et al, 2017 ). The testes can be alternatively fixed in a mixture of 3.7% formalin with 0.2% glutaraldehyde and 0.05% saponin ( Benson and Busch, 1996 ) or in Bouin solution ( Ellenburg et al, 2020 ). A wide range of other fixatives can be used for other tissue types and for special antibody staining (for reviews, see Hopwood, 1969 ; Arnold et al, 1996 ; Oliver and Jamur, 1999 ; Eltoum et al, 2001 ).…”

Rimklb mutation causes male infertility in mice

“…The tissues were perfused and additionally fixed using Bouin fixation 22 for 48 h. After fixation, the tissues were embedded in paraffin wax. Paraffin-embedded tissues were sliced to a thickness of six microns, attached to polylysine-coated slides, and dried at 40 °C overnight.…”