ZO-1 and F4/80 staining of the colon was detected by staining the colonic tissue sections (5 μm) with anti-ZO-1 antibody (Servicebio, GB11195, 1:200 dilution) in PBS, anti-F4/80 antibody (Servicebio, GB11027, 1:1000 dilution) in PBS and goat-anti-rabbit CY3 conjugated antibody (Servicebio, GB21303, 1:300 dilution) in PBS. Finally, the sections were counterstained with DAPI (Servicebio, G1012). At a temperature of − 18°C, 20 μm frozen brain sections were cut using a cryostat. The brain slices were blocked with 3% bovine serum albumin for 30 min at room temperature and then incubated with the primary antibodies at 4°C overnight. The primary antibody anti-Iba1 (Servicebio, GB13105, 1:500 dilution) and anti-GFAP (Servicebio, GB11096, 1:800 dilution) were used. After washing with PBS, the sections were incubated with the secondary antibodies at 37°C for 50 min. The secondary antibody goat-anti-rabbit Cy3 conjugated antibody (Servicebio, GB21303, 1:300 dilution) were used. Finally, the sections were counterstained with DAPI (Servicebio, G1012) and then imaged with microscope (Nikon Eclipse C1). Quantification of positively stained cells in the PFC and HIP regions were used ImageJ.
Gb21303
Manufactured by Wuhan Servicebio Technology
Sourced in China, United States
The GB21303 is a laboratory equipment designed for general scientific research purposes. It serves as an incubator, providing a controlled environment for various applications. The core function of the GB21303 is to maintain a stable temperature and humidity for samples or experiments that require a regulated environment.
Automatically generated - may contain errors
Lab products found in correlation
G1012, by Wuhan Servicebio Technology (14 mentions)
Eclipse c1, by Nikon (10 mentions)
Gb25303, by Wuhan Servicebio Technology (7 mentions)
Gb25301, by Wuhan Servicebio Technology (6 mentions)
Gb22303, by Wuhan Servicebio Technology (6 mentions)
Ds u3, by Nikon (6 mentions)
Cy3 conjugated goat anti rabbit igg h l, by Wuhan Servicebio Technology (4 mentions)
Cy3 conjugated goat anti rabbit igg, by Wuhan Servicebio Technology (4 mentions)
Gb21301, by Wuhan Servicebio Technology (4 mentions)
Gb113109, by Wuhan Servicebio Technology (4 mentions)
G5001, by Wuhan Servicebio Technology (4 mentions)
Gb113373, by Wuhan Servicebio Technology (4 mentions)
Gb22301, by Wuhan Servicebio Technology (4 mentions)
Lsm 880, by Zeiss (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Wuhan Servicebio Technology (4 mentions)
Lsm 700, by Zeiss (4 mentions)
Eclipse ti sr, by Nikon (3 mentions)
Gb23303, by Wuhan Servicebio Technology (3 mentions)
Gb11027, by Wuhan Servicebio Technology (3 mentions)
Gb12105, by Wuhan Servicebio Technology (3 mentions)
90 protocols using gb21303
1
Immunohistochemical Analysis of Colon and Brain
2
Immunofluorescence Staining of Mouse Lung Tumor Tissues
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Mouse lung tumor tissues were made into paraffin-embedded sections. For immunofluorescence staining, slides were fixed with 4% paraformaldehyde, permeabilized with 0.15% Triton X-100, blocked with 3% BSA for 30 min at room temperature (RT), and incubated with primary antibodies (F4/80, GB11027, Servicebio, 1:4000; CD24, DF8518, affinity, 1:100) in phosphate-buffered saline. Then, slides were incubated with secondary antibodies (Cy3-Goat anti-rabbit, GB21303, Servicebio, 1:300). For immunofluorescence double staining, slides were incubated with primary antibodies (F4/80, GB11027, Servicebio, 1:4000; CD86, DF6332, affinity, 1:200; CD206, GB13438, Servicebio, 1:500) in phosphate-buffered saline. Then, slides were incubated with secondary antibodies (HRP-Goat anti-rabbit, GB23303, Servicebio, 1:500; Cy3-Goat anti-rabbit, GB21303, Servicebio, 1:300) and FITC-Tyramide (G1222-50UL, servicebio). After staining, sections were placed under a microscope (Nikon Eclipse E100, Tokyo, Japan) for observation. Images were analyzed using Image-Pro Plus 6.0 software.
3
Expression and Localization of TL1A in BMDCs
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BMDCs were cultured as described above. After 7 d, BMDCs were washed with PBS and blocked with BSA blocking for 2 h. For the TL1A expression experiment, the BMDCs were incubated with primary antibodies for anti-TL1A (1:200, abcam, 85,566) for 16 h at 4 °C, and followed by PE-conjugated secondary antibodies (Servicebio, GB21303). For colocalization experiment, the BMDCs were incubated with primary antibodies for anti-TL1A (1:200, bioss, bs-5092R) and anti-RAF1 (1:400, proteintech, 66592-1-lg) for 16 h at 4 °C, and followed by FITC and PE-conjugated secondary antibodies (Servicebio, GB21303), respectively. After BMDCs were cytospin onto glass slides and stained by DAPI (Solarbio, S2110), sections were examined with a confocal microscope (Thermo).
4
Dual-Immunofluorescence Staining of Midbrain
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The procedures for double immunofluorescence staining were performed as that in our previous report [32 (link)]. In brief, free-floating 30-μm sections of the midbrain from each group were incubated with rabbit-derived anti-Iba1 (019-19741; 1:500; Wako Chemicals), rabbit anti-MEKK3 (NB100-92399; 1:100; Novus), and rabbit anti-TH (GB11181; 1:100; Servicebio) at 4 °C overnight followed by Cy3-conjugated goat anti-rabbit IgG (GB21303, 1:300; Servicebio) or a goat anti-mouse IgG conjugated with Alexa Fluor 488 (GB25301, 1:400; Servicebio) for 1 h at room temperature (22 ± 2 °C). Viewed under a LSM 880 (Carl Zeiss, Jena, Germany) laser scanning confocal microscopy, immunoreactivity exhibited green or red fluorescence. Confocal images were acquired and analysed using ZEN lite software (Carl Zeiss).
5
Immunofluorescence Assay of LC3A in MDBK Cells
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MDBK cells were seeded in 24 mm glass-bottomed microwell dishes. After was cultured according to the description mentioned above, MDBK cells were then washed with PBS three times and fixed with 4% paraformaldehyde (Servicebio) for 15 min. Next, 0.1% TritonX-100 (Servicebio) was used for cell permeabilization for 20 min. The cells were blocked with 3% BSA (Servicebio) in PBS at room temperature for 30 min and then were incubated with anti-LC3A (Novus Biologicals, NB100-2331, 1:100) overnight at 4℃. The next day, the cells were incubated with CY3 (Servicebio, GB21303, 1:300) at room temperature for 1h. The 4,6-diamidino-2-phenylindole (DAPI, Servicebio) counterstain was used to show the MDBK cells nuclei. Finally, the stained MDBK cells were observed by immunofluorescence microscopy (NIKON ECLIPSE C1).
6
Immunohistochemical Analysis of Protein Expression
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Immunohistochemistry analysis was conducted to evaluate the expression of relative proteins. The paraffin-embedded tissue sections were deparaffinized and treated with hydrogen peroxide (3 m/v) for 15 min to remove endogenous peroxidase. Antigen retrieval was performed by blocking the samples in goat serum for 10 min at 22°C. The following antibodies were added and incubated for 12 hours at 4°C: anti-TLR4 antibody (Servicebio, GB11519, 1:500), anti-LC3B antibody (Abcam, ab86714, 1:500), and anti-NF-κB p65 antibody (Servicebio, GB13025-1, 1:500). Cy3 (Servicebio, GB21303) and FITC (Servicebio, GB22303) secondary antibodies (1:250) were used for visualization. Tissue sections were mounted and analyzed using an inverted fluorescence microscope (Nikon Eclipse TI-SR) and imaging system (Nikon DS-U3).
7
Immunofluorescence Analysis of Colonic Markers
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Paraffin-embedded colonic mucosa sections first underwent deparaffinization and antigen retrieval and was then blocked with 5% BSA for 30 min. Then, the section was incubated with anti-PRLR (Abcam, ab170935, Cambridge, UK, 1: 250) and anti-TNFSF13B (Abcam, ab203791, 1: 100), and anti-CD11b (Abcam, ab52478, Cambridge, UK, 1: 250) at 4 °C overnight, with antibodies diluted in primary antibody dilution buffer (Servicebio, Wuhan, China). Subsequently, the tissues were washed by PBS for 3 times and incubated with fluorescent-labeled secondary antibody diluted in PBS at room temperature for 45 min (Servicebio, GB21303, 1:300; GB25303, 1: 400). Finally, DAPI staining was conducted at room temperature for 20 min. Fluorescence microscope (Olympus IX73, Tokyo, Japan) was used to observe the fluorescent images.
8
Immunostaining of p16INK4a in Paraffin Sections
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Paraffin sections were dewaxed in xylene and rehydrated in ethanol and distilled water. After antigen retrieval with EDTA, sections were blocked in 3% BSA for 30 min at room temperature. The blocked solution was gently shaken off and the primary antibody, namely, Anti -CDKN2A/p16INK4a Rabbit pAb) (GB111143, Servicebio, Wuhan, China) was added to the sections and incubated overnight at 4°C. The next day, a secondary antibody (Cy3 conjugated Goat Anti-Rabbit IgG, GB21303, Servicebio, Wuhan, China) was added and incubated at room temperature for 1 h. After DAPI (G1012, Servicebio, Wuhan, China) stained the nuclei, the sections were dried slightly and then sealed with anti-fluorescence quenched sealing tablets. Images of the cortex were captured at ×20 magnification with a fluorescence microscope (Nikon DS-U3, Nikon).
9
Immunofluorescence Assay for H9C2 Cells
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After instilling different treatment regimens, the H9C2 cells were fixed for 30 min with 4% paraformaldehyde, rinsed three times with PBS, and treated for 20 min with 0.2% Triton X‐100 (Solarbio) at room temperature, followed by another regimen of three times of PBS rinses. Next, the H9C2 cells were blocked using 5% BSA at room temperature for 30 min and incubated with corresponding antibodies at 4°C overnight. The corresponding antibodies were against LC3B (dilution ratio of 1:200, 3868, Cell Signaling Technology, Beverly, MA, USA), noligomerization domain‐like receptor protein (NLRP3) (dilution ratio of 1: 200, NBP2‐12446, NovusBio, CO, USA), and caspase‐1(dilution ratio of 1: 200, ab1872, Abcam). Next, the H9C2 cells were incubated with Cy3‐conjugated goat anti‐rabbit IgG (H + L) (dilution ratio of 1: 300, GB21303, Servicebio, Wuhan, Hubei, China) at 37°C for 1 h. Afterwards, the H9C2 cells were stained with 4′,6‐diamidino‐2‐phenylindole (Solarbio) for 5 min and observed under a fluorescence microscope (Olympus).
10
Immunofluorescence Analysis of Pyroptosis Pathway
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Mouse corneal sections and HCECs on 6-chamber slides were fixed with 4% paraformaldehyde (Solarbio, #P1110) and permeabilized with 0.2% Triton X-100 (Sigma–Aldrich, #9036-19-5) at room temperature for 10 min. The samples were then incubated with NLRP3 (1:50, Novus Biologicals, #NBP2-12446SS), ASC (1:50, Santa Cruz Biotechnology, #sc-514414), CASP1 (1:200, ABclonal, #A0964), GSDMD (1:100, ABclonal, #A18281) and Ly-6G antibodies (1:200, Servicebio, #GB11229) at 4°C overnight, followed by incubation at room temperature with secondary antibodies (1:300, Servicebio, #GB21303) for 1 h. For TdT-mediated dUTP nick end labeling (TUNEL) staining, the sections were incubated with TUNEL dye (Beyotime, #1086) for 30 min under shade. Nuclei were labeled with DAPI for 10 min. Finally, the samples were observed and photographed with a Leica TCS SP5 confocal microscope (Leica, Germany). For further detection the pyroptotic cells in corneal tissues, double-immunofluorescence staining of active CASP1 and TUNEL was performed on corneal sections. Active CASP1+/TUNEL+ cells were determined as pyroptotic cells (24 (link)).
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