Immunohistochemical staining was conducted based on the procedures in our previous report [4 (link)]. Specifically, the overnight incubation (4 °C) of testicular tissue sections was executed by virtue of the rabbit polyclonal antibody against ZO-1 (diluted at 1:100; bs-1329R; Bioss) or LC3 (1:100 dilution; 14600-1-AP, Proteintech) in combination with the mouse monoclonal SOX9 antibody (diluted at 1:100; ab76997; abcam, an SC-specific marker). Later, the tissue sections, washed 3 times in PBS, were cultured for 45 min using the FITC-coupled goat anti-rabbit IgG (H+L) antibody (1:200 dilution; HS111, TransGen, Beijing, China) and the PE-labeled goat anti-mouse IgG (H+L) antibody (1:200 dilution; HS221, TransGen, Beijing, China). Lastly, the cell nuclei received DAPI staining based on the previously described methods, followed by observation and photography of the sections under the Olympus-DP73 optical microscope (Tokyo, Japan).
Dp73 optical microscope
Manufactured by Olympus
Sourced in Japan
The Olympus-DP73 is a high-resolution digital optical microscope. It features a 12.8-megapixel camera for capturing detailed images of specimens. The microscope provides precise magnification and focusing capabilities to enable clear visualization of samples.
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9 protocols using dp73 optical microscope
1
Immunohistochemical Analysis of Testicular Tissue
2
Uterine Tissue Histological Staining
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Uterine tissues were fixed in 4% (v/v) paraformaldehyde with 0.1 M PBS (pH 7.4) and embedded in paraffin. Sections of 4 μm thickness were mounted onto gelatin/poly L-lysine-coated glass slides and dried in an incubator at 60 °C for 2 h. Paraffin sections were submerged in hematoxylin dyeing solution (D10393; Bioss, Beijing, China) for 2 min, 1% Acid Alcohol Fast Differentiation Solution (C0163M; Beyotime, Haimen, China) for 3 s, and eosin (S0159; Bioss) for 1 min. The sections were then processed through gradient alcohol and xylene dehydration. Sections were observed and photographed using an Olympus-DP73 optical microscope (Olympus, Tokyo, Japan).
3
Histological Analysis of Testicular Tissues
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The porcine and murine testicular tissues embedded in paraffin were made into 4 μm thick sections for HE staining. The Olympus-DP73 optical microscope (Tokyo, Japan) was employed to assess the dynamic changes in the histology of the testes.
4
Histological Analysis of Testicular Morphology
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Testes fixed in 4% PFA solution (pH 7.3) were processed using standard histological procedures. Briefly, testes were cut into blocks (0.5–1 mm3 each) and embedded in paraffin wax. Paraffin sections (4 μm) were dewaxed in dimethyl benzene and then processed with decreasing alcohol gradient. Samples were then treated in haematoxylin, hydrochloric acid, eosin, and increasing alcohol gradient, then finally in xylene. After processing, samples on glass slides were covered with mounting medium (neutral resin), then cover slides, and viewed under Olympus-DP73 optical microscope (Tokyo, Japan) for photographic images. Testicular morphology and structural differences among tissues were observed and compared.
The organ (testicular) index was determined as follows: Organ (testicular) index = organ weight/body weight × 100.
The organ (testicular) index was determined as follows: Organ (testicular) index = organ weight/body weight × 100.
5
Histological Analysis of Abomasal Tissue
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The fixed tissue samples were processed using standard histological procedures. Briefly, they were cut into blocks (1cm3 each) and embedded in paraffin wax. Paraffin sections (4μm) were cut, de-waxed in dimethyl-benzene, and then processed with decreasing alcohol gradient. The samples were then treated in haematoxylin, hydrochloric acid, eosin and with increasing alcohol gradient, and finally in xylene. After processing, the specimens were mounted on glass slides with embedding medium (neutral resin) and then covered with coverslips and viewed under Olympus-DP73 optical microscope (Tokyo, Japan) for photographic images. The histological features of the abomasal tissue, including the reticular and linear parts were carefully investigated in each group.
6
Immunohistochemical Analysis of Ngb and HIF-1α in Yak and Cattle Telencephalon
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Tissue samples from different regions of yak and cattle telencephalon were dehydrated, embedded with paraffin, and cut into 4-μm-thick sections, hematoxylin-eosin (HE) routine staining. The sections were rehydrated, repaired with 0.125% trypsin antigen, blocked with 5% goat serum, and incubated at 37°C with the primary antibody of rabbit anti-Ngb, anti-HIF-1α polyclonal antibody (1:400, Bioss, China, bs-1859R and bs-0737R). The sections were then incubated with the secondary antibody, and then added with streptomyces avidin-peroxidase solution. The immunoperoxidase color reaction was developed with the HRP-DAB substrate chromogen solution. The labeled samples were then counterstained with hematoxylin. Negative controls were performed by substituting the primary antibody with PBS (0.01mol/L, pH =7.2). Then the sections were observed and photographed with an Olympus-DP73 optical microscope (Tokyo, Japan).
7
Immunofluorescent Detection of PGR in Sheep Follicular GCs
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PGR expression in sheep follicular GCs was detected using immunofluorescence. Cultured GCs were fixed in 4% (v/v) paraformaldehyde, washed with 4 °C PBS, treated with 0.1% (v/v) Triton X-100 for 30 min, and then incubated with 5% (w/v) bovine serum albumin (BSA) for 30 min. The cells were then mixed with polyclonal rabbit anti-PGR (1:300, bs-23376R, Bioss, Beijing, China) and with 2% BSA (negative control). Detection of PGR antibody with fluorescein isothiocyanate (FITC)-coupled goat anti-mouse immunoglobulin (IgG) (H+L) antibody (1:200, TransGen, Beijing, China), was then treated with 1 μg/ml 4,6 diamidino 2 phenyl indole (4’6’-diamindino-2-phenylindole, Solarbio, Beijing, China), and was used for nuclear reverse staining. Digital image acquisition was performed with an Olympus DP73 optical microscope (Olympus, Tokyo, Japan).
8
TUNEL Assay for Apoptosis Detection
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Excised tumor tissues were fixed in 4% paraformaldehyde, paraffin embedded, and sectioned. Some sections were stained with hematoxylin and eosin (H&E) for histological examination following the standard protocol, and some sections were subjected to TUNEL assay. TUNEL staining was performed with the In Situ Cell Death Detection Kit (Roche, Basel, Switzerland). Sections were permeabilized with 0.1% v/v Triton-X 100 for 8 min at RT. Endogenous peroxidase was blocked by incubating the sections with 2% H2O2 for 10 min. Sections were incubated with the enzyme solution and TUNEL reaction solution for 1 h at 37°C, followed by incubation with Converter-POD for 30 min at 37°C. Thereafter, Diaminobenzidine (DAB) (Solarbio) was added for a brief incubation of 3 to 5 min followed by counterstaining with hematoxylin for the nuclei. The sections were mounted and observed under an Olympus DP73 optical microscope.
9
Colon Tissue Histological Analysis
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We separated colon tissue of four groups of rats (n = 9 each group) and dissected the colon along the longitudinal axis of intestine. The most damaged colon tissue (2 cm) was fixed in 4% paraformaldehyde for 24 h at -80°C and then embedded in paraffin. The sections were 5 μm thick, stained with hematoxylin-eosin (H&E) and periodic acid-Schiff (PAS), and observed under a DP73 optical microscope (Olympus, Tokyo, Japan). Colon integrity, changes in colonic recesses, goblet cell distribution, and inflammatory cell infiltration were all observed and photographed.
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