Seven-day-old rats (P7) were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg) and fixed in situ by intracardiac perfusion with 4% paraformaldehyde. The whole brain was removed and subsequently embedded in paraffin for tissue sectioning. The 5 µm paraffin sections were dewaxed and brough into water through a graded ethanol series. Antigen retrieval was performed in 0.01 M citrate buffer (pH 6.0) in a microwave oven. The sections were blocked with goat serum and incubated for 12 h at 4°C with primary antibody (Rat mAb to MBP, 1:200 dilution, Abcam AB7349; Rb pAb to NG2, 1:100 dilution, Abcam ab129051). The sections were then washed in PBS, incubated at room temperature for 1 h with the secondary antibody (HRP Goat Anti-Rat IgG, 1:400 Dilution, Abbkine A21040; HRP Goat Anti-Rabbit IgG 1:400 Dilution, Abbkine A21020), and washed again. After diaminobenzidine (DAB) and hematoxylin staining, the nuclei were observed under a light microscope.
Hrp goat anti rabbit igg
Manufactured by Abbkine
Sourced in United States
HRP Goat Anti-Rabbit IgG is a secondary antibody conjugated with Horseradish Peroxidase (HRP). It is designed to detect and bind to rabbit primary antibodies in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab129051, by Abcam (1 mentions)
Ab7349, by Abcam (1 mentions)
Lysis buffer, by Merck Group (1 mentions)
Anti occludin, by Cell Signaling Technology (1 mentions)
Anti bax, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence kit, by PerkinElmer (1 mentions)
Anti claudin 3, by Cell Signaling Technology (1 mentions)
Sds page, by Ipsen (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Keygen Biotech (1 mentions)
Rack1, by Cell Signaling Technology (1 mentions)
O glcnac, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg hrp, by Abbkine (1 mentions)
Myc tag, by Cell Signaling Technology (1 mentions)
Pd l1, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
5 protocols using hrp goat anti rabbit igg
1
Immunohistochemical Localization of MBP and NG2 in Rat Brain
2
Western Blot Analysis of Cellular Proteins
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Ileal tissues and liver tissues were lysed using a lysis buffer (Sigma, USA). Total protein concentration was determined using the BCA method. Western blotting analysis was performed as previously described [44 (link)]. The primary antibodies included rabbit anti-IκBα, anti-GAPDH, anti-p38, anti-phospho-p38 (p-p38), anti-JNK, anti-phospho-JNK (p-JNK), anti-extracellular signal-regulated kinase (anti-ERK), anti-p-ERK, anti-Atg5, anti-Beclin 1, anti-SOD2, anti-Bax, anti-claudin-3, anti-occludin, anti-zonula occludens 1 (ZO-1), anti-LC3 (Cell Signaling Technology, USA; 1:1000). The second antibody was HRP, goat anti-rabbit IgG (Abbkine, Beijing; 1:5000).
Specific proteins were detected using an enhanced chemiluminescence kit (Perkin Elmer Life Sciences, USA). Protein bands were visualized with a chemiluminescence substrate and a gel-imaging system (Tanon Science and Technology, shanghai) and analyzed with Image Analysis software (NIH, USA). In all instances, the density values of bands were corrected after subtracting background values. GAPDH was used as the internal reference protein.
Specific proteins were detected using an enhanced chemiluminescence kit (Perkin Elmer Life Sciences, USA). Protein bands were visualized with a chemiluminescence substrate and a gel-imaging system (Tanon Science and Technology, shanghai) and analyzed with Image Analysis software (NIH, USA). In all instances, the density values of bands were corrected after subtracting background values. GAPDH was used as the internal reference protein.
3
Western Blot Analysis of MTDH, GPX4, and β-Actin
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Cell lysates were lysed with RIPA Lysis Buffer (KeyGEN, China) and cocktail (MCE, USA). After boiling, the lysate separation was performed with 10% SDS-PAGE (EpiZyme, China). Then transferred PVDF membranes (Millipore, USA) were blocked with 5% milk and incubated with indicated antibodies at 4°C overnight. The antibodies used in this study are listed: MTDH (Proteintech, 13 860-1-AP, 1:500), β-actin (CST, 8457 s, 1:1000), GPX4 (CST, 52455, 1:1000), and HRP Goat Anti-rabbit IgG (Abbkine, A21020, 1:10 000). The membranes were detected using ECL detection reagents (KeyGEN).
4
Quantifying Inflammatory Markers in Rat Joints
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Western blotting (WB) was performed to detect the expressions of NLRP3, caspase-1, and IL-1β proteins in joint grinds from the rat model. Protein lysates were extracted using RIPA buffer (Epizyme Biomedical PC101) and protease inhibitor (Epizyme Biomedical GRF101). Electrophoresis was performed under a segmented constant voltage (stacking gel, 80 V; voltage gel, 120 V). The bands were transferred to a membrane in an ice bath with a constant current (200 mA). The membrane was then blocked with 5% skim milk powder for 1 h. The following antibodies were used for probing overnight at 4°C: IL-1β (1 : 1000, CST D3A3Z), caspase-1 (1 : 1000, CST D7F10), NLRP3 (1 : 1000, CST D4D8T), and GAPDH (1 : 1000, CST D16H11). The membrane was then incubated with goat anti-rabbit IgG-HRP (1 : 5000, Abbkine A21010) for 1 h at room temperature. The results were scanned using chemiluminescence detection with TanonFine-DoX6 (Tianneng).
5
Protein Expression Analysis of SBSGL Treated Cells
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Cells were cultured in six‐well plates with SBSGL at 0, 600, 1200 and 1800 μg/mL concentrations. Radio immunoprecipitation assay buffer (Shanghai Biyuntian Biotechnology Co., lot no. P0013C) with a protease inhibitor was used to extract proteins. Equal amounts of protein from each sample were transferred to polyvinylidene fluoride membranes (Merck, lot no. IPFL00010). Membranes were incubated with primary antibodies against RACK1 (Cell Signaling Technology, lot no. 5432, 1:1000), OGT (Cell Signaling Technology, lot no. 24083, 1:1000), O‐GlcNAc (Thermo Fisher Scientific, lot no. MA1‐072, 1:1000), LC3B (Cell Signaling Technology, lot no. 3868, 1:1000), p62 (Cell Signaling Technology, lot no. 5114, 1:1000), PD‐L1 (Cell Signaling Technology, lot no. 13684, 1:1000), Myc‐tag (Cell Signaling Technology, lot no. 2276, 1:1000) and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH, Cell Signaling Technology, lot no. 5174, 1:1000) overnight at 4°C. After washing with Tris‐buffered saline with Tween (TBST, Biosharp, lot no. 21259419), secondary antibodies goat anti‐rabbit IgG‐HRP (Abbkine, lot no. A21020, 1:10,000) and goat anti‐mouse IgG‐HRP (Abbkine, lot no. A21010, 1:10,000) were applied for 1 h at room temperature. Membranes were washed thrice with TBST, and visualization was achieved using an ECL luminescence kit (Shanghai Biyuntian Biotechnology Co., lot no. P0018S).
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