The tumor tissues were fixed in 4% paraformaldehyde buffered with phosphate-buffered saline for 24 h at room temperature, embedded in paraffin and serially sectioned (4 µm). Next, the tissue sections were blocked in 5% goat serum (Wuhan Servicebio Technology Co., Ltd.) for 15 min at room temperature, and incubated with primary DUSP4 antibody (1:100; cat. no. ab72593; Abcam) at 4°C overnight. The sections were incubated with HRP-conjugated anti-rabbit (1:200; cat. no. GB23303; Wuhan Servicebio Technology Co., Ltd.) at 37°C for 20 min, and then visualized using a PV-9000 DAB detection kit according to the manufacturer's protocol. The sections were counterstained with hematoxylin for 3 min at room temperature and observed under a light microscope (Olympus Corporation; scale bar, 100 µm). DUSP4 staining was graded semi-quantitatively as previously described (16 (link)). Staining intensity was graded as 1 (no staining), 2 (weak staining), 3 (clear staining), or 4 (strong staining). The IHC images regarding Grade 1–4 tumour tissues are presented in Fig. 1 . The intensity and abundance (expressed as a fraction) were multiplied to obtain the total immunoreactivity score.
Goat serum
Manufactured by Wuhan Servicebio Technology
Sourced in China
Goat serum is a biological fluid derived from the blood of goats. It contains a complex mixture of proteins, hormones, and other biomolecules that are essential for cellular functions. The core function of goat serum is to provide a nutrient-rich environment for cell culture and various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscope, by Zeiss (2 mentions)
Cy3 conjugated goat anti mouse igg, by Wuhan Servicebio Technology (2 mentions)
Fitc conjugated goat anti rabbit igg, by Wuhan Servicebio Technology (2 mentions)
Anti cbs rabbit pab, by Wuhan Servicebio Technology (2 mentions)
Anti c fos mouse pab, by Wuhan Servicebio Technology (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Southern Biotech (1 mentions)
Triton x 100, by Solarbio (1 mentions)
Optical microscope, by Olympus (1 mentions)
Kim 1, by Abcam (1 mentions)
Exosome depleted fbs, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Wuhan Servicebio Technology (1 mentions)
Phosphate buffered saline (pbs), by Wuhan Servicebio Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Wuhan Servicebio Technology (1 mentions)
Fv1200, by Olympus (1 mentions)
Pontamine sky blue, by Merck Group (1 mentions)
D ap5, by Merck Group (1 mentions)
Capsazepine, by Merck Group (1 mentions)
L703606, by Merck Group (1 mentions)
7 protocols using goat serum
1
Immunohistochemical Analysis of DUSP4 in Tumor Tissues
2
Immunohistochemical Analysis of Ovarian Proliferation
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Firstly, the paraffin-embedded ovarian sections (5-µm-thick) underwent antigen retrieval using sodium citrate buffer (Wuhan Servicebio Technology Co., Ltd.). Subsequently, permeabilization was performed using 0.3% Triton X-100 (Beijing Solarbio Science & Technology Co., Ltd.) and blocking was performed with 5% goat serum (Wuhan Servicebio Technology Co., Ltd.). The slides were then incubated overnight at 4°C with primary antibodies, including Ki67 (1:600; cat. no. GB111141-100, Wuhan Servicebio Technology Co., Ltd.) and proliferating cell nuclear antigen (PCNA; 1:600; cat. no. GB11010-100, Wuhan Servicebio Technology Co., Ltd.). The following day, the slides were incubated with the secondary FITC-conjugated antibody (1:400; cat. no. GB22303, Wuhan Servicebio Technology Co., Ltd.) and Cy3-conjugated antibody (1:400; cat. no. GB21303, Wuhan Servicebio Technology Co., Ltd.) for 1 h. Finally, the cell nuclei were stained with DAPI for 10 min at room temperature (SouthernBiotech), and the slides were observed under a fluorescence microscope (Carl Zeiss AG).
3
Immunohistochemical Analysis of Kidney Injury
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For immunohistochemical staining, 10% buffered formalin-fixed at room temperature for 24 h, and paraffin-embedded tissue sections (4 µm) were blocked with 10% goat serum (Wuhan Servicebio Technology Co., Ltd.) for 2 h at room temperature and were incubated with primary antibodies against F4/80 (1:200; product code ab6640; Abcam) and kidney injury molecule-1 (KIM-1) (1:200; cat. no. MA5-28211; Invitrogen; Thermo Fisher Scientific, Inc.) for 12 h at 4°C and then analyzed using a streptavidin peroxidase detection system (50 µl; cat. no. KIT-9720; Fuzhou Maixin Biotech Co., Ltd.) at room temperature according to the manufacturer's protocol. DAB (Fuzhou Maixin Biotech Co., Ltd.) was used as an HRP-specific substrate. The images were visualized under an optical microscope (Olympus Corporation).
4
Identification of Human Ovarian Granulosa Cells
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The human ovarian granulosa cell line, HO-23 (37 (link),38 (link)), was obtained from Qingqi (Shanghai) Biotechnology Development Co., Ltd. (cat. no. BFN60808800) and cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% exosome-depleted FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C and 5% CO2.
The immunofluorescence staining of follicle-stimulating hormone (FSH) receptor (FSHR) was performed to identify the human GCs (hGCs), as FSHR is specifically expressed in the cytoplasm of ovarian GCs. Initially, the complete medium was removed, and the cells were washed three times with PBS (Wuhan Servicebio Technology Co., Ltd.). Subsequently, permeabilization was achieved using 0.3% Triton X-100 (Wuhan Servicebio Technology Co., Ltd.), followed by blocking with 5% goat serum (Wuhan Servicebio Technology Co., Ltd.). The cells were then incubated overnight at 4°C with primary antibody against FSHR (1:800; cat. no. GB11275-1-100, Wuhan Servicebio Technology Co., Ltd.). On the following day, the cells were incubated with a FITC-conjugated secondary antibody (1:400; cat. no. GB22303, Wuhan Servicebio Technology Co., Ltd.) for 1 h. Finally, the cell nuclei were stained with DAPI for 10 min at room temperature (cat. No.: 0100-20, SouthernBiotech). Images were captured using a ZEISS fluorescence microscope (Carl Zeiss AG).
The immunofluorescence staining of follicle-stimulating hormone (FSH) receptor (FSHR) was performed to identify the human GCs (hGCs), as FSHR is specifically expressed in the cytoplasm of ovarian GCs. Initially, the complete medium was removed, and the cells were washed three times with PBS (Wuhan Servicebio Technology Co., Ltd.). Subsequently, permeabilization was achieved using 0.3% Triton X-100 (Wuhan Servicebio Technology Co., Ltd.), followed by blocking with 5% goat serum (Wuhan Servicebio Technology Co., Ltd.). The cells were then incubated overnight at 4°C with primary antibody against FSHR (1:800; cat. no. GB11275-1-100, Wuhan Servicebio Technology Co., Ltd.). On the following day, the cells were incubated with a FITC-conjugated secondary antibody (1:400; cat. no. GB22303, Wuhan Servicebio Technology Co., Ltd.) for 1 h. Finally, the cell nuclei were stained with DAPI for 10 min at room temperature (cat. No.: 0100-20, SouthernBiotech). Images were captured using a ZEISS fluorescence microscope (Carl Zeiss AG).
5
Immunofluorescence Staining Protocol
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Cells were washed 3 times with PBS and fixed with 4% paraformaldehyde for 15 min. Coverslips were then rinsed with 0.5% Triton 100× in PBS for 10 min after washing with PBS. After being blocked with goat serum (Servicebio Technology) for 30 min at room temperature, cells were incubated with primary antibody overnight at 4°C. Alexa Fluor 488-conjugated secondary antibodies were used for visualization. The nuclei were labeled with DAPI for 5 min. Images were acquired with a laser scanning confocal microscopy (FV1200, Olympus Corp., Tokyo, Japan).
6
Quantification of Hydrogen Sulfide Signaling
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NaHS (2, 4, and 8 nmol), PDTC (2 nmol), D-AP5 (2 nmol), and pontamine sky blue were all purchased from Sigma-Aldrich (St. Louis, MO, United States). NaHS was dissolved in 0.9% normal saline, while the other chemicals were dissolved in dimethyl sulfoxide. Goat serum, anti-CBS rabbit pAb, FITC-conjugated goat anti-rabbit IgG, Cy3 conjugated Goat anti-mouse IgG, and anti-c-Fos mouse pAb were purchased from Servicebio.
7
Hydrogen Sulfide Signaling Pathway Study
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NaHS, L703606, PDTC, Capsazepine, and protamine sky blue were purchased from Sigma-Aldrich (St. Louis, MO, United States). NaHS was dissolved in 0.9% saline, while the other chemicals were dissolved in dimethyl sulfoxide and reconstituted in saline. For the immunohistochemical fluorescence double labeling, the following reagents were purchased from Servicebio (Wuhan, China): Goat serum, anti-CBS rabbit pAb, FITC-conjugated goat anti-rabbit IgG, Cy3-conjugated goat anti-mouse IgG, and anti-c-Fos mouse pAb.
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