Brain tissues (n = 6) were put into 30% sucrose solution for 24 h and cut into 30 μm coronal sections using a Leica CM1950 cryostat (Leica Microsystems). After washing with PBS, coronal sections were sealed with 10% goat serum (C0265, Beyotime) and then incubated with primary antibodies of Drp1 (ab184247, Abcam) and Parkin (JF82-09, Novus). The sections were then sealed with 10% goat serum for 1 h and then incubated with Tomm20 antibody (H00009804-M01, Abnova). Thereafter, the sections were stained with fluorochrome-conjugated secondary antibody (ZF-0311, ZSGB-BIO), glial fibrillary acidic protein antibody (1 : 5000; ab7260, Abcam), and ionized calcium-binding adapter molecule 1 antibody (1 : 500; 019-19741, Wako). In brief, paraffin sections were immunostained with anti-nuclei (NeuN) antibody (ab177487, Abcam) at 4°C overnight and subsequently subjected to terminal deoxynucleotidyl transferase dUTP nick-end labeling staining using an In Situ Cell Death Detection kit (Cat. No. 11 684 795 910) according to the manufacturer's protocol. Finally, the sections were incubated with 4′,6-diamidino-2-phenylindole (C1002, Beyotime) nuclear dye, and the sections were visualized using a confocal laser scanning microscope (FV1000, Olympus). Sections from forebrain, midbrain, and hindbrain regions were randomly selected, and the ischemic penumbra was counted for statistical analysis.
C0265
Manufactured by Beyotime
Sourced in China
C0265 is a laboratory equipment product. It is a general-purpose device used for various scientific applications in research and testing environments.
Automatically generated - may contain errors
Lab products found in correlation
Fv1000, by Olympus (2 mentions)
C1002, by Beyotime (2 mentions)
Ab6721, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Goat serum, by Beyotime (2 mentions)
Ab184247, by Abcam (1 mentions)
Ab177487, by Abcam (1 mentions)
Ab7260, by Abcam (1 mentions)
In situ cell death detection kit, by Abcam (1 mentions)
Leica cm1950, by Leica Microsystems (1 mentions)
Zf 0311, by ZSGB-BIO (1 mentions)
P0081, by Beyotime (1 mentions)
Ab11370, by Abcam (1 mentions)
Ab150080, by Abcam (1 mentions)
P0099, by Beyotime (1 mentions)
Trixton x 100, by Beyotime (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
H 1200, by Vector Laboratories (1 mentions)
N storm, by Nikon (1 mentions)
Rabbit anti mouse igg h l, by Abcam (1 mentions)
14 protocols using c0265
1
Immunohistochemical Analysis of Brain Tissue
2
Quantifying Myocardial Angiogenesis Post-Infarction
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Myocardium from the infarct and border regions was obtained 4 weeks after gene transfection and immunohistochemical staining for von Willebrand Factor (vWF) was performed. Sections of rabbit myocardium from the infarcted and border regions were fixed with 4% paraformaldehyde at room temperature for 24 h and embedded in paraffin. The sections were incubated in a citrate buffer (0.01 M; pH 6.0; P0081; Beyotime Institute of Biotechnology) at 100°C for 15 min for antigen retrieval. The sections were then blocked in blocking buffer (5% normal goat serum; C0265; Beyotime Institute of Biotechnology) at 37°C for 20 min and incubated with a rabbit anti-vWF antibody (1:200; bs-10048R, Beijing Biosynthesis Biotechnology Co., Ltd.) overnight at 4°C. Sections were subsequently incubated with biotin conjugated secondary antibody (goat anti-rabbit IgG; 1:200; bs-0295G-Bio, Beijing Biosynthesis Biotechnology Co., Ltd.) for 40 min and treated with streptavidin peroxidase for 10 min at 37°C Finally, specimens were incubated in DAB for 5 mins at 37°C. The microvascular density (MVD) was then determined using optical microscopy (x200) according to the Weidner criteria (23 (link)). A pathologist was employed to average the five visual fields with the most capillaries in single-blind manner.
3
Immunohistochemical Analysis of Cx43 Expression
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Six hours after exposure, the round coverslips were placed in a 24-well plate, and 1 mL of 4% paraformaldehyde (P0099, Beyotime, Shanghai, China) was added to each well for 15 min; 1 mL of permeabilization buffer with Trixton-X-100 (P0096, Beyotime, Shanghai, China) was added to rupture the membrane for 10 min; 1 mL of 10% goat serum (diluted 1:10 in PBS; C0265, Beyotime, Shanghai, China) was used as blocking solution at room temperature for 1 h. Then, 200 μL of Cx43 primary antibody (1:100 dilution in 10% goat serum; ab11370, Abcam, Cambridge, UK) was added and incubated for 1 h at room temperature; then, fluorescently labeled secondary antibody (1:100 dilution in 10% goat serum; ab150080, Abcam, Cambridge, UK) was added and incubated in the dark at room temperature for 1 h. The round coverslips were removed and put on slides, and after the DAPI mounting medium (H-1200, Vector Laboratories, Newark, CA, USA) was added, the square coverslips were covered and fixed with nail polish. The images were observed under a fluorescence microscope (Nikon, Tokyo, Japan). The optical density values were semi-quantified, and statistical analysis was conducted. The cells were washed with DPBS (14190144, Gibco, Carlsbad, CA, USA) 3 times, 3 min each time, before each medium change.
4
Collagen-II Expression in HNPCs
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In the light of a previous delineation, the expression of Collagen-II was observed through ICC [30 (link)]. The treated HNPCs were collected and washed with PBS, and then fixed with 4% paraformaldehyde at room temperature for 15 min. Next, cells were treated with 0.2% of Triton X-100 (P0096, Beyotime, Shanghai, China) for 10 min, and incubated firstly with 10% goat serum (C0265, Beyotime, Shanghai, China) for 20 min, and then with the diluted primary antibody (Collagen II Monoclonal Antibody; MA1-37,493, Thermo Fisher Scientific, Waltham, Massachusetts, USA) at 4°C overnight. Subsequently, cells were incubated with the diluted secondary antibody (Rabbit Anti-Mouse IgG H&L; ab6728, Abcam, Cambridge, UK) at room temperature for 1 h. Later, the cells were treated with DAB working solution (P0202, Beyotime, Shanghai, China), and then washed with distilled water. Lastly, the Collagen-II content of cells was observed (under 200 × magnification) using fluorescence microscope (N-STORM, Nikon, Tokyo, Japan).
5
Immunohistochemical Analysis of Caveolin-1 and Collagen I
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After antigen retrieval in water bath and normal goat serum (C0265, Beyotime) blocking, the slides were incubated with primary antibodies to Caveolin-1 (ab32577, 1:2000, Abcam) or collagen I (ab270993, 1:500, Abcam) (Fang et al. 2018 (link)), which was followed by incubation with secondary antibody goat anti-rabbit IgG (ab6721, 1:1000, Abcam). Finally, the staining processes were performed with diaminobenzidine colorimetric reagent solution (P0203, Beyotime) and hematoxylin (C0107, Beyotime). HE staining was used for histological observation as previously described (Liu et al. 2021 (link)).
6
Immunohistochemical Analysis of HCC and Tumors
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Paraffin sections of human HCC tissue samples and mouse tumor tissues were dewaxed, dehydrated in ascending series of alcohol and subjected to antigen retrieval in a water bath. Next, the sections were blocked with normal goat serum (C0265, Beyotime) for 20 min, and immunostained with primary rabbit antibodies to CD38 (ab216343, 1:1000, Abcam) and Ki67 (ab15580, 1:200, Abcam) at 4°C overnight and with secondary antibody of goat anti-rabbit IgG (ab6721, 1:1000, Abcam) at 37°C for 20 min. Subsequently, the sections were observed under a microscope in 5 randomly selected high-power fields from each section. The results were evaluated by an experienced pathologist. The positive rate was calculated: positive rate = positive cells/total cells.
7
Immunofluorescence Staining of Mouse Skin
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Mouse skin tissues were fixed with 4% paraformaldehyde for overnight. Then, the tissues were washed, sealed with normal saline of 0.01 M phosphate buffer for three times, and then sealed with 10% goat serum (C0265, Beyotime, Shanghai, China) at room temperature for 30 min. After that, transforming growth factor-β1 (TGF-β1) (1:200, ab92486, Abcam), vascular endothelial growth factor (VEGF) (1:200, ab2350, Abcam), and platelet-derived growth factor (PDGF-BB) (1:200, ab9704, Abcam) were incubated with tissues at 4 °C for overnight. The secondary antibody and 4′,6-diamidino-2-phenylindole were incubated at room temperature in the dark for 1 h, and glycerol was fixed. The confocal laser scanning microscope was used for analysis (LSM, FV1000; Olympus Corp., Tokyo, Japan).
8
Immunofluorescence Analysis of FSHR and AMH
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HGCs were
cultured on coverslips (14 mm, NEST) at 5 × 104 cells/mL
in 6-well plates for 2 days. Then, the coverslips were washed twice
with PBS and fixed in 4% paraformaldehyde (PFA). After protein blocking
(C0265, Beyotime, China) for 30 min at 37 °C, the coverslips
were incubated with an antibody against follicle-stimulating hormone
receptor (FSHR, 1:100, 22665-1-AP, Proteintech, China) and an antibody
against anti-Müllerian hormone (AMH, 1:100, 23479-1-AP, Proteintech)
diluted in PBS overnight at 4 °C. The next day, all the coverslips
were washed and incubated with secondary antibodies (1:500, Cy3-labeled
goat antirabbit IgG, A0516, Beyotime) at room temperature for 2 h.
4′,6′-Diamidino-2-phenylindole (DAPI, C1005, Beyotime)
was used to visualize nuclei. Images were observed and captured using
an Olympus IX73 inverted microscope (Olympus, China).
cultured on coverslips (14 mm, NEST) at 5 × 104 cells/mL
in 6-well plates for 2 days. Then, the coverslips were washed twice
with PBS and fixed in 4% paraformaldehyde (PFA). After protein blocking
(C0265, Beyotime, China) for 30 min at 37 °C, the coverslips
were incubated with an antibody against follicle-stimulating hormone
receptor (FSHR, 1:100, 22665-1-AP, Proteintech, China) and an antibody
against anti-Müllerian hormone (AMH, 1:100, 23479-1-AP, Proteintech)
diluted in PBS overnight at 4 °C. The next day, all the coverslips
were washed and incubated with secondary antibodies (1:500, Cy3-labeled
goat antirabbit IgG, A0516, Beyotime) at room temperature for 2 h.
4′,6′-Diamidino-2-phenylindole (DAPI, C1005, Beyotime)
was used to visualize nuclei. Images were observed and captured using
an Olympus IX73 inverted microscope (Olympus, China).
9
Immunofluorescence Staining Protocol
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Followed by three cycles of washing with PBS, 5 min each, 0.5% TritonX-100 was provided dropwise to slides and let stand by for 10 min at room temperature. The slides were immersed in PBS 3 times, 3 min each, blocked by using 10% normal goat serum (C0265; Beyotime) at room temperature, and removed after blocking for 60 min. The primary antibody diluent (P0262; Beyotime) was supplemented, placed in a humid chamber, and incubated at 4°C overnight. The slides were rewarmed at room temperature for 30 min and soaked in PBS for 3 times, 3 min each. Excess liquid was removed, and fluorescent secondary antibody was diluted and added for incubation at 37 °C for 60 min. PBS immersion was conducted for 3 times again, 3 min each. Following the addition of DAPI (C1005; Beyotime), the cells were incubated in the dark for 5 min and stained for nucleus. Antifluorescence quenching medium was added to mount the slides, and the experimental results were collected using fluorescence microscope imaging system (MF53; Guangzhou Mshot Photoelectronic Technology Co., Ltd.).
10
Neuronal Cell Identification by Immunofluorescence
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We identified the extracted neuronal cells by immunofluorescence staining experiments. The extracted cells were washed twice with PBS and then fixed with 4% paraformaldehyde at 4°C for 20 min. After washing again, the cells were perforated with 0.1% Triton X-100 (4°C, 15 min, V900502-100ML, Sigma, St. Louis, MO, USA). After washing, the culture dish was blocked by adding 5% goat serum (C0265, Beyotime, Nantong, China). After 30 min, Neuronal specific enolase (NSE) primary antibody (1:200, ab220216, Abcam, Camb, UK) was added to the cells overnight (4°C). After the incubation, FITC-labeled goat anti-rabbit secondary antibody (AS-28176-1-FITC, Anaspec, Fremont, CA, USA) was used for immunostaining. After 2 hr, the nuclei of hippocampal neurons were stained with 4',6-diamidino-2-phenylindole (DAPI, 10236276001, Roche, Basel, Switzerland) for 5 min. After thorough washing with PBS, the anti-fluorescence quenching capsule (E675011-5ml, BBI, Shanghai, China) and neutral gum were used to seal the tablets. The positive expression of NSE was obtained by fluorescence microscopy.
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