Histological sections (5 μM) were deparaffinized and rehydrated. Antigen retrieval was performed by heating the slides in a 10 mM citrate buffer in a pressure cooker. Subsequently, the specimens were washed in 0.01 M phosphate-buffered saline (PBS), treated with 3% hydrogen peroxide, and washed in 0.01 M PBS again. Blocking was performed with 3% BSA in 0.01 M PBS, and then the specimens were incubated with the respective reagents overnight at 4°C. For the detection of the expression of IκBα protein, anti-IκBα polyclonal IgG antibody (1:200; Santa Cruz Biotechnology) was used as a primary antibody, and donkey antirabbit IgG–HRP antibody (1:200; Santa Cruz Biotechnology) was used as a secondary antibody. Thereafter, these slides were stained with diaminobenzidine (Beyotime, Haimen, China) and counterstained with hematoxylin. Sections 5 μm thick containing both tumor and normal tissues taken from each patient were used for IκBα immunohistochemistry.
Donkey anti rabbit igg hrp antibody
Manufactured by Santa Cruz Biotechnology
Sourced in Germany
The Donkey anti-rabbit IgG-HRP antibody 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
Sheep anti mouse igg hrp antibody, by Merck Group (3 mentions)
Rabbit anti rat aqp3 antibody, by Alomone (2 mentions)
Diaminobenzidine, by Beyotime (1 mentions)
Elisa kit, by R&D Systems (1 mentions)
Ecl prime detection reagent, by GE Healthcare (1 mentions)
Imagequant las 500 system, by GE Healthcare (1 mentions)
Mouse anti rabbit glyceraldehyde 3 phosphate dehydrogenase gapdh antibody, by Merck Group (1 mentions)
High capacity cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Gefitinib, by Fujifilm (1 mentions)
Water soluble tetrazolium salt wst 1, by Roche (1 mentions)
Alexa fluor 488 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Erlotinib, by LC Laboratories (1 mentions)
Imagequant las 500, by Cytiva (1 mentions)
5 protocols using donkey anti rabbit igg hrp antibody
1
Immunohistochemical Analysis of IκBα Expression
2
Quantification of Fibrosis-Related Cytokines in Liver Tissue
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One hundred milligrams of liver tissues was homogenized with RIPA buffer and centrifuged at 10,000 ×g for 15 min at 4°C. The supernatant fraction was used to determine the levels of fibrosis-related cytokines. The protein levels of TGF-β1, PDGF-BB, and tissue inhibitor of matrix metalloprotease (TIMP) in the liver tissue were also determined using an ELISA kit (R&D Systems, Minneapolis, MN). The quantification of CTGF was performed using a modification of the sandwich ELISA method described previously [23 (link)]. Briefly, a 96-well ELISA plate was coated with 100 mL of goat polyclonal anti-rat antibody at a concentration of 10 mg/mL in PBS and 0.02% sodium azide overnight. After incubation with blocking buffer (PBS, 0.02% sodium azide and 1% bovine serum albumin) and washing (four times), 50 mL of the sample or recombinant human CTGF standard was added for 1 hour. Then, 100 mL of the primary rabbit polyclonal anti-goat antibody (2 mg/mL) and 50 mL of the secondary donkey anti-rabbit IgG-HRP antibody (both 1 : 2000) were added (Santa Cruz Biotechnology, Germany). CTGF was quantified by measuring the absorbance at 405 nm after mixing the substrate solution and stopping solution (2 N H2SO4).
3
Quantitative Immunoblotting of AQP3 Protein
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Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane. The proteins in the membrane were probed with primary (rabbit anti-rat AQP3 antibody, 1/500, Alomone Labs, Jerusalem, Israel; mouse anti-rabbit GAPDH antibody, 1/10,000, Merck Millipore, Darmstadt, Germany) and secondary antibodies (donkey anti-rabbit IgG-HRP antibody, 1/5000, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; sheep anti-mouse IgG-HRP antibody, 1/10,000, Merck Millipore). The recognized proteins were detected using an ECL prime detection reagent (GE Healthcare, Chicago, IL, USA). The protein immunocomplexes were visualized using a Lumino image analyzer (ImageQuant LAS500 system, GE Healthcare). The protein expression level of AQP3 was normalized to that of GAPDH.
4
Molecular Mechanisms of EGFR Signaling
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Erlotinib was purchased from LC Laboratories (Woburn, MA, USA). Gefitinib was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). TRI reagent was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). A high-capacity cDNA synthesis kit was purchased from Applied Biosystems (Foster City, CA, USA). The rabbit anti-rat aquaporin-3 (AQP3) antibody was purchased from Alomone Labs (Jerusalem, Israel). The mouse anti-rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody and sheep anti-mouse IgG-HRP antibody were purchased from Merck Millipore (Darmstadt, Germany). The donkey anti-rabbit IgG-HRP antibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The rabbit anti-human EGFR antibody, rabbit anti-human phospho (p)-EGFR antibody, rabbit anti-human p44/42 mitogen-activated protein kinase (MAPK) antibody, and rabbit anti-human p-p44/42 MAPK antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). Alexa Fluor 488 donkey anti-rabbit IgG was purchased from Thermo Fisher Scientific (Waltham, MA, USA). An enhanced chemiluminescence (ECL) prime detection reagent was purchased from GE Healthcare (Chicago, IL, USA). The cell proliferation reagent water-soluble tetrazolium salt (WST-1) was purchased from Roche (Mannheim, Germany).
5
Quantifying Membrane Protein Levels
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After measuring the protein concentration of PM fraction, western blotting was performed. After blocking the poly vinylidene difluoride membrane, it was treated with primary antibodies, rabbit anti‐rat AQP3 antibody (Alomone Labs, Jerusalem, Israel) or mouse anti‐Na+/K+ ATPase α‐1 antibody (Merck Millipore, Darmstadt, Germany). Secondary antibodies used were sheep anti‐mouse IgG‐HRP antibody (Merck Millipore) or donkey anti‐rabbit IgG‐HRP antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After the membrane was treated with detection reagents, and the bands were analyzed by luminoimage analyzer ImageQuant LAS500 (Cytiva, Tokyo, Japan).
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