Methylmercuric chloride (MeHgCl; Sigma-Aldrich) was dissolved in PBS to form a stock concentration of ; MG132; 3,4-Dihydroxybenzoic acid (DHB); cobalt chloride ( ); N-acetyl-cysteine (NAC) and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid); and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl diphenyltetrazolium bromide (MTT)] were obtained from Sigma-Aldrich. Cycloheximide (CHX) was purchased from MedChemExpress; 2-methoxyestradiol (2-MeOE2) was purchased from Selleck Chemicals. For Western blotting analysis, the primary polyclonal antibodies to , VEGF-A, GLUT-1, and EPO were obtained from ImmunoWay, and antibodies to and were obtained from Cell Signaling Technology and CMATAG, respectively. Secondary antibodies used for immunoblotting included horseradish peroxidase (HRP)-conjugated antimouse (Santa Cruz Biotechnology) or anti-rabbit antibodies (Santa Cruz Biotechnology).
Anti rabbit antibodies
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-rabbit antibodies are immunoglobulin molecules produced by the immune system of animals in response to the introduction of rabbit-derived proteins. These antibodies are designed to specifically recognize and bind to rabbit-derived molecules, facilitating their detection, purification, or neutralization in various experimental and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Cobalt chloride, by Merck Group (1 mentions)
Methylmercuric chloride mehgcl, by Merck Group (1 mentions)
2 methoxyestradiol 2 meoe2, by Selleck Chemicals (1 mentions)
Mg132, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
N acetylcysteine, by Merck Group (1 mentions)
Trolox, by Merck Group (1 mentions)
3 4 dihydroxybenzoic acid, by Merck Group (1 mentions)
Cycloheximide (chx), by MedChemExpress (1 mentions)
Anti ki67 antibody, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Ecl kit, by Abcam (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Cell line nucleofector kit r, by Lonza (1 mentions)
Enhanced chemiluminescence ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
M per extraction reagent, by Thermo Fisher Scientific (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Tripure reagent, by Roche (1 mentions)
6 protocols using anti rabbit antibodies
1
Hypoxia-Inducible Factor Regulation
2
Immunohistochemical Staining of Histone Modifications
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Consecutive sections of tissue samples were deparaffinized with xylene, rehydrated with alcohol, and subjected to the immunohistochemical staining of intermittent wave irradiation as previously described [36 (link)]. The rabbit anti-acetyl-histone-3 (Lys 9/14), anti-acetyl-histone 4 (Lys 8), and anti-ki-67 antibody and anti-rabbit antibodies conjugated to HRP were purchased from Santa Cruz Biotechnology and Dako respectively. Negative controls were prepared by omitting the primary antibody.
Two independent observers (LH and ZHC) randomly selected and counted 100 cells from five representative fields from each section. Any discrepancies were checked by both observers until a consensus was reached. Positive expression was graded as follows: 0 = negative; 1 = 1%–50%; 2 = 50%–74%; 3 ≥ 75%. The staining intensity was graded as follows: 1 = weak; 2 = intermediate; 3 = strong. The two grades were multiplied to obtain a final score: – = 0; + = 1–2; ++ = 3–5; +++ = 6–9).
Two independent observers (LH and ZHC) randomly selected and counted 100 cells from five representative fields from each section. Any discrepancies were checked by both observers until a consensus was reached. Positive expression was graded as follows: 0 = negative; 1 = 1%–50%; 2 = 50%–74%; 3 ≥ 75%. The staining intensity was graded as follows: 1 = weak; 2 = intermediate; 3 = strong. The two grades were multiplied to obtain a final score: – = 0; + = 1–2; ++ = 3–5; +++ = 6–9).
3
Hippocampal Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
The right frozen hippocampi were homogenized with 200 µL lysis buffer [Ripa buffer and inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA), 1:20] for 1 hour and centrifuged at 12000 g (4°C) for 20 minutes. Protein concentration was determined with a Bio-Rad assay system (Bio-Rad, San Francisco, CA, USA), and 100 micrograms of total protein from each sample were denatured with sample buffer (6.205 mM tris-HCl, 10% glycerol, 2% SDS, 0.01% bromophenol blue and 50 mM 2-ME) at 95°C for 5 minutes. The denatured proteins were separated on a SDS page (10% sodium dodecyl sulphate polyacrylamide gel) and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Non- specific bindings were blocked with 5% nonfat dry milk, and membranes were probed with anti-BDNF, -NT4, Trk-β (1:500; Santa Cruz, CA, USA), and β-actin (1:1000; Sigma Aldrich, St. Louis, MO, USA) monoclonal antibodies for 2 hours and secondary anti- rabbit antibodies (1:5000; Santa Cruz, CA, USA) conjugated to alkaline phosphatase (for Trk-β) or horseradish peroxidase (for BDNF and NT-4) for 1 hour. Bands were detected using 5-bromo-4-chloro-3-indolyl phosphate in the presence of nitroblue tetrazolium (Abcam, Cambridge, UK) or ECL kit (Abcam, Cambridge, UK) as a chemiluminescent substrate. Band densities were measured by an image analysis system (UVIdoc, Houston, TX, USA).
+ Expand
The right frozen hippocampi were homogenized with 200 µL lysis buffer [Ripa buffer and inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA), 1:20] for 1 hour and centrifuged at 12000 g (4°C) for 20 minutes. Protein concentration was determined with a Bio-Rad assay system (Bio-Rad, San Francisco, CA, USA), and 100 micrograms of total protein from each sample were denatured with sample buffer (6.205 mM tris-HCl, 10% glycerol, 2% SDS, 0.01% bromophenol blue and 50 mM 2-ME) at 95°C for 5 minutes. The denatured proteins were separated on a SDS page (10% sodium dodecyl sulphate polyacrylamide gel) and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Non- specific bindings were blocked with 5% nonfat dry milk, and membranes were probed with anti-BDNF, -NT4, Trk-β (1:500; Santa Cruz, CA, USA), and β-actin (1:1000; Sigma Aldrich, St. Louis, MO, USA) monoclonal antibodies for 2 hours and secondary anti- rabbit antibodies (1:5000; Santa Cruz, CA, USA) conjugated to alkaline phosphatase (for Trk-β) or horseradish peroxidase (for BDNF and NT-4) for 1 hour. Bands were detected using 5-bromo-4-chloro-3-indolyl phosphate in the presence of nitroblue tetrazolium (Abcam, Cambridge, UK) or ECL kit (Abcam, Cambridge, UK) as a chemiluminescent substrate. Band densities were measured by an image analysis system (UVIdoc, Houston, TX, USA).
4
Histological Evaluation of Cartilage Grafts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For histology and immunohistochemistry, the cartilage grafts were fixed in 4% paraformaldehyde, dehydrated with a series of graded alcohol, embedded in paraffin, and 6 μm thick sections were cut. The sections were stained with HE for morphology and lympholeukocyte infiltration analysis and toluidine blue for sulfated glycosaminoglycans. Immunohistochemistry was performed for aggrecan and collagen II using primary, polyclonal anti-rabbit antibodies (1:100; Santa Cruz). Sections were deparaffinized, digested with methanol and 0.1% Triton X-100 for membrane permeability, and washed with phosphate buffered saline (PBS). Then, the sections were treated with 3% hydrogen peroxide at 37°C for 30 minutes to inactivate the endogenous peroxidases, incubated with normal goat serum for 30 minutes to block nonspecific binding, and incubated with 1:200 mouse anti-rat collagen II or goat anti-rat aggrecan primary antibody overnight at 4°C. After washing in PBS, the sections were incubated with horseradish-conjugated anti-mouse or anti-goat IgG secondary antibodies for 30 minutes at room temperature. The signal was developed using 3, 3′-diaminobenzidine (DAB kit; Zhongshan Biotechnology Inc., Beiijng, People’s Republic of China). The negative control was performed without incubation of the primary antibody.
5
Protein Expression and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All reagents were obtained from Sigma-Aldrich unless stated otherwise. The tissue culture reagents, including McCoy’s 5A and DMEM medium, fetal bovine serum (FBS), were from Invitrogen (Eggenstein, Germany). Cell Line Nucleofector®Kit R was from Lonza (Allendale, NJ, USA). TriPure Reagent, LightCycler 480 SYBR Green I Master PCR reaction mix, PhosStop phosphatase inhibitor, and cOmplete Protease Inhibitor Cocktail were from the Roche Diagnostics Corporation (Indianapolis, ID, USA). All primers were made by Genomed (Warsaw, Poland). M-MLV Reverse Transcriptase was purchased from Promega Corp. (Madison, WI, USA). Enhanced Chemiluminescence (ECL) Western blotting substrate, M-PER Extraction Reagents, NE-PER Nuclear and the BCA Protein Assay Kit, Moloney Murine Leukemia Virus Reverse Transcriptase were from Thermo Scientific Pierce (Minneapolis, MN, USA). Rabbit anti-vinculin antibodies were from Cell Signaling (Danvers, MA, USA), goat anti-mouse antibodies, anti-rabbit antibodies, and mouse anti-GAPDH conjugated with horseradish peroxidase were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA).
6
Western Blot Analysis of ER Stress Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extracts from HL7702 cells or mice livers were prepared in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). Protein concentration was determined by the BCA assay (Beyotime Biotechnology). Then, equal amounts of protein (about 50 μg) were subjected to SDS-PAGE and transferred to a PVDF membrane. The membranes were blocked in a 5% BSA-or skim milk-containing TBST buffer (Tris-HCl, pH 7.5, 140 mM NaCl and 1% Tween 20) for 1 hr at room temperature, and then probed with specific primary anti-Bip antibody (1:1000), anti-CHOP (1:1000), anti-phospho-eIF2α (1:1000) or β-actin antibody (1:2000) overnight. The PVDF membranes were then incubated with HRP-conjugated antimouse (1:20000, CWBIO, Beijing, China) or anti-rabbit antibodies (1:20000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The bands on the membranes were visualized by chemiluminescence detection (Thermo Scientific, Rockford, IL, USA) and quantitated using Quantity One Software (Bio-Rad).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!