Epididymides were fixed in Bouin’s fluid (Polysciences) at 4°C overnight. Fixed epididymides were dehydrated by increasing ethanol concentrations, and then were embedded with paraffin. Paraffin sections (5-μm) were stained with Mayer hematoxylin solution for 3 to 5 minutes, counterstained with eosin Y solution [53% (v/v) ethanol, 0.3% (v/v) eosin, and 0.5% (v/v) acetic acid] for 2 to 5 minutes, dehydrated in increasing ethanol concentrations, and finally mounted in Permount or Entellan new (Merck). The caudal epididymal spermatozoa were observed with phase contrast microscopy.
Permount
Manufactured by Merck Group
Sourced in United States, Germany, Japan
Permount is a mounting medium used for the preparation of permanent microscope slides. It is a fast-drying, clear, and colorless solution that is commonly used to mount and preserve biological specimens for microscopic examination. Permount provides a refractive index that is suitable for a wide range of applications in the field of microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Avidin biotin peroxidase complex solution, by Vector Laboratories (9 mentions)
Hematoxylin, by Merck Group (7 mentions)
Bx51 light microscope, by Olympus (7 mentions)
Bluing solution, by Thermo Fisher Scientific (4 mentions)
Cryostat, by Leica (3 mentions)
Nanozoomer, by Hamamatsu Photonics (3 mentions)
Vectastain abc kit, by Vector Laboratories (3 mentions)
Cryomicrotome, by Leica (3 mentions)
Entellan new, by Merck Group (2 mentions)
Bouin s fluid, by Polysciences (2 mentions)
Fluorescence latex beads, by Merck Group (2 mentions)
Diff quik staining dye, by Merck Group (2 mentions)
3 3 diaminobenzidine (dab), by Merck Group (2 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (2 mentions)
Picrosirius red, by Abcam (2 mentions)
Eosin solution, by Thermo Fisher Scientific (2 mentions)
Cresyl violet solution, by Merck Group (2 mentions)
Biotinylated goat anti rabbit secondary antibody, by Jackson ImmunoResearch (2 mentions)
Rabbit anti fos, by Santa Cruz Biotechnology (2 mentions)
Histoclear, by Merck Group (2 mentions)
84 protocols using permount
1
Histological Analysis of Epididymal Sperm
2
FFPE Liver Immunostaining for LC3B
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After deparaffinization and rehydration of FFPE liver sections according to a standard protocol, antigen retrieval was conducted by boiling in 10 mM sodium citrate buffer. After cooling down, the slides were permaebilized and blocked using 0.4% Triton X-100 (BioBasic) and 5% BSA (Capricorn) in PBS, consecutively. After that, the slides were incubated with an anti-LC3B antibody (Thermo Fisher Scientific) overnight at 4 °C. After twice washing with PBS (10 minutes per wash), the slides were further incubated with a Dylight 488 conjugated anti-rabbit secondary antibody (Thermo Fisher Scientific) for 2 hours in the dark at room temperature. Finally, the slides were washed and mounted by using Permount (Merck) for further analysis under fluorescence microscope.
3
Histological Analysis of Liver Sections
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Liver sections (5 μm) were first deparaffinized and rehydrated according to the procedure mentioned above. The sections were subjected to consecutive staining with hematoxylin and eosin, respectively. The sections were dehydrated in absolute ethanol and xylene, respectively, and were then mounted with Permount (Merck). Finally, the sections were observed with an inverted microscope.
4
Histological Analysis of Epididymal Sperm
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Epididymides were fixed in Bouin's fluid (Polysciences, Warrington, PA, USA) at 4 °C overnight. Fixed epididymides were dehydrated by increasing ethanol concentrations and then were embedded with paraffin. Paraffin sections (5‐μm) were stained with Mayer hematoxylin solution for 3 to 5 min, counterstained with eosin Y solution [53% (v/v) ethanol, 0.3% (v/v) eosin, and 0.5% (v/v) acetic acid] for 2 to 5 min, dehydrated in increasing ethanol concentrations, and finally mounted in Permount or Entellan new (Merck, Kenilworth, NJ, USA). The caudal epididymal spermatozoa were observed with phase contrast microscopy.
5
Immunohistochemical Analysis of Endometrial Cancer
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Human endometrial cancer tissue microarray slides containing 97 cases of EC patients and 5 normal uterus tissues were purchased from TissueArray.Com LLC (Derwood, MD, USA. Cat. No. EM1021c); for these microarrays, tissue collection was performed with informed consent from the donors. After dewaxing and antigen retrieval, the slides were incubated with each antibody, including anti-EXOSC5 (GeneTex Inc., Hsinchu City, Taiwan. Catalog No. GTX118473), anti-NTN4 (Sigma-Aldrich, Catalog No. HPA049832), anti-phospho-FAKTyr397 (ABclonal, Inc., Woburn, MA, USA. Catalog No. AP0302), or anti-c-MYC (Santa Cruz Biotechnology Inc., Dallas, TX, USA. Catalog No. sc-40), followed by signal development with a standard avidin-biotin-peroxidase complex method with 3,3'-diaminobenzidine substrates (DAKO, Carpinteria, CA). Negative controls included serial sections from which either the primary or secondary antibodies were excluded. The slides were counterstained with haematoxylin and mounted with Permount (Merck, Darmstadt, Germany), and the images were captured and analysed with a TissueFAXS Plus Microscopy System (TissueGnostics GmbH, Vienna, Austria).
6
Phagocytic Activity Evaluation Protocol
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The phagocytosis was calculated according to (Yoshida, Kitao, 1991) with slight modification, for detail see (Van Doan, Hoseinifar, Sringarm, Jaturasitha, Yuangsoi, Dawood, Esteban, Ringø, Faggio, 2019) . Briefly, 200µL of leucocyte cell suspensions ( 2x 10 6 cells mL -1 ) were loaded on coverslips and incubated at room temperature for two hours. After incubation, the coverslips were washed with 3mL of RPMI-1640 to remove any non-adherent cells. Then, a solution of 200µL of fluorescence latex beads with a concentration of 2 x 10 7 of beads (mL -1 ) (Sigma-Aldrich, USA) was placed into each coverslip and incubated again at room temperature for 1.5 hours. The coverslips were then rewashed with 3mL of RPMI-1640 to remove any non-phagocytized bead. After washing, the coverslips were then fixed with methanol, and stained with Diff-Quik staining dye (Sigma-Aldrich, USA) for ten seconds. After staining, a wash of PBS (pH 7.4) removed any excessive stains. The washed coverslips were allowed to dry at room temperature and then attached to the slides with Permount (Merck, Germany). The number of phagocyte cells per 300 adhered cells was later counted microscopically. The phagocytic index (PI) and phagocytic rate (PR%) were calculated through the following equations: PI = (Number of phagocytized beads divided by the number of phagocytizing leukocytes) *100.
7
Immunohistochemical Staining Protocol
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Paraffin sections (5 μm) were deparaffinized and incubated with the primary antibodies (1:100 dilution) using a standard avidin–biotin–peroxidase complex method. The slides were incubated with 3,3′-diaminobenzidine (DAKO, Carpinteria, CA, USA) to detect the antibody binding. The slides were then counterstained with hematoxylin, mounted with Permount (Merck, Darmstadt, Germany), and scanned/analyzed with a TissueFAXS PLUS system (TissueGnostics, Vienna, Austria).
8
Immunohistochemical Analysis of Cell Markers
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Histological sections were placed on organosilane-pretreated slides, deparaffinized with xylene, rehydrated, and incubated with 3% hydrogen peroxide for 5 min to block endogenous peroxidase activity. For different cells quantification, the slides were incubated with primary antibodies for 2 h: monoclonal mouse anti-human α-SMA antibody (1A4 clone, code M0851, 1:250 dilution, Dako), polyclonal goat anti-rat MCP-1 antibody (code R17, 1:100 dilution, Santa Cruz Biotechnology Inc.), or polyclonal goat anti-human TGF-β1 antibody (code 6G, 1:300 dilution, Santa Cruz Biotechnology Inc.). Subsequently, the sections were incubated with a second biotin antibody (Universal Kit, Novocastra Laboratories Ltd.) for 15 min. Then, the histological sections were developed using 3,3′-diaminobenzidine (Sigma Chemical Co.) as a chromogen and light counterstaining with Meyer hematoxylin. The slides were dehydrated in graded alcohol, cleared in xylene, and mounted in Permount (Merck, Darmstadt, Germany). We also prepared negative controls, omitting the primary antibody from the assay and replacing it with nonimmune goat serum for MCP-1 and TGF-β1 and nonimmune mouse serum for α-SMA.
9
Immunohistochemical Analysis of Tight Junction Proteins
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To proceed with IHC staining, colonic tissue was immersed in 4% paraformaldehyde at 4°C for 2~4 h and post-fixation was performed. The tissue was stored in 30% sucrose, which completely penetrated the tissue, and then cut with a cryostat (Leica) to a thickness of 10 μm. IHC was subsequently performed. To check the extent to which tight junction proteins appear, rabbit polyclonal anti-Zo-1 IgG, rabbit polyclonal anti-occludin IgG, and rabbit polyclonal anti-claudin IgG were reacted with the primary antibody. Next, tissue sections were incubated with the secondary antibody and avidin and biotinylated peroxidase complex. The color development of the antibody was induced with 3,3'-diaminobenzidine (DAB, Merck, Germany) using an ABC kit (Vector Laboratories, USA). After drying, the samples were sealed in Permount (Merck) using ethanol and xylene, and observed using an optical microscope (BX60, Olympus Co., Japan).
10
Phagocytic Activity Evaluation Protocol
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The phagocytosis was calculated according to (Yoshida, Kitao, 1991) with slight modification, for detail see (Van Doan, Hoseinifar, Sringarm, Jaturasitha, Yuangsoi, Dawood, Esteban, Ringø, Faggio, 2019) . Briefly, 200µL of leucocyte cell suspensions ( 2x 10 6 cells mL -1 ) were loaded on coverslips and incubated at room temperature for two hours. After incubation, the coverslips were washed with 3mL of RPMI-1640 to remove any non-adherent cells. Then, a solution of 200µL of fluorescence latex beads with a concentration of 2 x 10 7 of beads (mL -1 ) (Sigma-Aldrich, USA) was placed into each coverslip and incubated again at room temperature for 1.5 hours. The coverslips were then rewashed with 3mL of RPMI-1640 to remove any non-phagocytized bead. After washing, the coverslips were then fixed with methanol, and stained with Diff-Quik staining dye (Sigma-Aldrich, USA) for ten seconds. After staining, a wash of PBS (pH 7.4) removed any excessive stains. The washed coverslips were allowed to dry at room temperature and then attached to the slides with Permount (Merck, Germany). The number of phagocyte cells per 300 adhered cells was later counted microscopically. The phagocytic index (PI) and phagocytic rate (PR%) were calculated through the following equations: PI = (Number of phagocytized beads divided by the number of phagocytizing leukocytes) *100.
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