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Published on: January 5, 2015
Characterization and quenching of autofluorescence in piglet testis tissue and cells
1Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, SK, Canada S7N 5B4.
Insights
Piglet testes exhibit intrinsic fluorescence that can hinder cell identification. Sudan Black B effectively blocks this autofluorescence, enabling clear visualization of gonocytes for research.
Area of Science:
- Reproductive Biology
- Cell Biology
- Histology
Background:
- Intrinsic fluorescence in biological tissues can impede accurate cell identification using fluorescence microscopy.
- Autofluorescence from granules within piglet testis interstitial cells was observed, potentially interfering with fluorescence-based assays.
Purpose of the Study:
- To investigate the characteristics of intrinsic fluorescence in piglet testis tissue and cells.
- To evaluate the efficacy of Sudan Black B in masking autofluorescence without affecting specific cell labeling.
Main Methods:
- Epifluorescence microscopy, confocal laser scanning microscopy, and flow cytometry were used to detect intrinsic fluorescence.
- Piglet testis tissue and cells were treated with Sudan Black B for varying incubation times.
- Specific fluorescence labeling of gonocytes was performed using Dolichos biflorus agglutinin (DBA) lectin.
Main Results:
- Granules within piglet testis interstitial cells exhibited broad autofluorescence emission (425-700 nm).
- Sudan Black B treatment effectively masked autofluorescence in 10-15 minutes for tissue sections and 8 minutes for cells.
- Specific gonocyte labeling with DBA remained unaffected after Sudan Black B treatment.
Conclusions:
- Lipofuscin or similar pigments in Leydig cell granules are likely responsible for piglet testis autofluorescence.
- Sudan Black B is a reliable method for blocking autofluorescence in piglet testes.
- This technique enhances the identification and characterization of piglet gonocytes via fluorescence microscopy.
Abstract:
Significant intrinsic fluorescence in tissues and in disassociated cells can interfere with fluorescence identification of target cells. The objectives of the present study were (1) to examine an intrinsic fluorescence we observed in both the piglet testis tissue and cells and (2) to test an effective method to block the autofluorescence. We observed that a number of granules within the testis interstitial cells were inherently fluorescent, detectable using epifluorescence microscopy, confocal laser scanning microscopy, and flow cytometry. The emission wavelength of the autofluorescent substance ranged from 425 to 700 nm, a range sufficiently broad that could potentially interfere with fluorescence techniques. When we treated the samples with Sudan Black B for different incubation times, the intrinsic fluorescence was completely masked after treatment for 10-15 min of the testis tissue sections or for 8 min of the testis cells, without compromising specific fluorescence labeling of gonocytes with lectin Dolichos biflorus agglutinin (DBA). We speculate that the lipofuscin or lipofuscin-like pigments within Leydig cell granules were mainly responsible for the observed intrinsic fluorescence in piglet testes. The method described in the present study can facilitate the identification and characterization of piglet gonocytes using fluorescence microscopy.
