Alpha-actin smooth muscle (α-SMA) is known as a specific marker widely expressed in fibroblasts [6 (link)]. After one week when CAFs were completely extracted from the tissue, they were cultured in a 25 cm² flask for one more week and then were stained on Day 14. The anti-α-SMA antibody (1/500, Abcam (Cambridge, UK), Cat. No.: ab5694) and HRP-conjugated secondary goat anti-rabbit IgG (1/2000, Abcam (Cambridge, UK), Cat. No.: ab205718) were used to identify CAFs by immunocytochemistry based on the company instructions on Day 14.
Hrp conjugated secondary goat anti rabbit igg
Manufactured by Abcam
Sourced in United Kingdom
HRP-conjugated secondary goat anti-rabbit IgG is a laboratory reagent used for the detection of rabbit primary antibodies in various immunoassays. It consists of goat-derived secondary antibodies that are conjugated to the enzyme horseradish peroxidase (HRP). This conjugation allows for the amplification and visualization of the target rabbit antibody signal.
Automatically generated - may contain errors
Lab products found in correlation
Anti α sma antibody, by Abcam (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Bcl 2, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Immobilon ecl substrate kit, by Merck Group (1 mentions)
Biospectrum 600 imaging system, by Analytik Jena (1 mentions)
Anti sox11, by Abcam (1 mentions)
Fetal bovine serum (fbs), by Bioidea (1 mentions)
Cdna synthesis kit, by Biofact (1 mentions)
Dulbecco modified eagle medium (dmem), by Bioidea (1 mentions)
Ecl plus detection reagent, by Cytiva (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Image analysis software, by Bio-Rad (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Tris glycine sds, by Bio-Rad (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
Gradient precast protein gel, by Bio-Rad (1 mentions)
5 protocols using hrp conjugated secondary goat anti rabbit igg
1
Identification of Cancer-Associated Fibroblasts
2
Western Blot Analysis of Cell Signaling Proteins
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Cell lysates were prepared with RIPA buffer (Thermo Scientific). The concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Pierce, Thermo Scientific). Immunoreactive bands were detected by using the Immobilon ECL substrate kit (Millipore, Merck KGaA, Germany). The images were acquired by using BioSpectrum 600 Imaging System (UVP, CA, USA). Antibodies used included primary and secondary antibodies, primary antibodies including anti-SOX11 (1:1000, Abcam), Bcl-2 (1:500, Abcam), Bax (1:500, Abcam), Cleaved-caspase3 (Anti-active Caspase-3, 1:500, Abcam) and anti-GAPDH (1:10000, Abcam); secondary antibody was HRP-conjugated secondary goat anti-rabbit IgG (1:2000, Abcam).
3
Isolation and Characterization of Cancer Stem Cells
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The following materials were used in this study: RPMI 1640 media (Bio Idea, Tehran, Iran, Cat. No.: BI-1006), fetal bovine serum (FBS) (Bio Idea, Tehran, Iran, Cat. No.: BI-1201), phosphate-buffered saline (PBS; Bio Idea, Tehran, Iran, Cat. No.: BI-1401), DMEM (Bio Idea, Tehran, Iran, Cat. No.: BI-1012), anti-α-SMA antibody (1/500, Abcam (Cambridge, UK), Cat. No.: ab5694), HRP-conjugated secondary goat anti-rabbit IgG (1/2000, Abcam, Cat. No.: ab205718), 24-well 8.0 μm pore size Cell Culture plate (Corning, Cat. No.: 353097), human LIF protein (Sino Biological, Beijing, China, Cat. No.: LIF 14890-HNAH), LIF receptor inhibitor antibody (Millipore (Burlington, MA, USA), Cat. No.: MABD150), FITC-conjugated mouse monoclonal antibody against human CD44 (5/100, Biolegend(San Diego, CA, USA), Cat. No.: 338803), PE-conjugated mouse monoclonal antibody against human CD24 (5/100, Biolegend, (San Diego, CA, USA), Cat. No.: 311105), Total RNA Kit (Yekta Tajhiz, Tehran, Iran, Cat. No.: YT9080), and cDNA synthesis kit (BioFact, Daejeon, Korea, Cat. No.: BR441-096).
4
Protein Expression Analysis in Tissues
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Tissue samples collected were homogenized in ice-cold tissue lysate buffer and proteins were extracted according to the manufacturer’s instructions (Beyotime, Haimen, China). The total protein concentration of the supernatant was determined using the BCA protein assay kit (Pierce Chemical Co., Rockford, IL). One hundred micrograms of proteins were loaded on SDS-PAGE (Bio-Rad, Laboratories, Inc., California) and transferred onto a nitrocellulose membrane (Bio-Rad, Laboratories, Inc., California). The membrane was blocked at 37 C for 2 h with 5% nonfat dry milk in TBS-T (Tris-buffered saline containing 0.1% Tween-20). AMHRII and INSR were detected using primary polyclonal rabbit anti-AMHRII (Abcam, Cambridge, UK) followed by secondary HRP-conjugated goat anti-rabbit IgG (Abcam, Cambridge, UK) and primary monoclonal mouse anti-INSR (NeoMarkers, California, USA) followed by secondary HRP-labeled anti-mouse IgG (Jackson ImmunoResearch Laboratories, Bar Harbor, ME), respectively. The bands were visualized using the ECL Plus detection reagent (Amersham Pharmacia Biotech, Piscataway, NJ), and then Band densities were quantified with the image analysis software (Bio-Rad, Laboratories, Inc., California).
5
SDS-PAGE and Western Blotting Protocol
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SDS–PAGE was conducted using the standard protocol65 (link). The samples were denatured for 5 min at 95 °C in 1× Laemmli sample buffer (Bio-Rad) before loading on a 4–15% gradient precast protein gel (Bio-Rad). The electrophoresis was performed at a constant voltage of 200 V in SDS running buffer (Tris/Glycine/SDS, Bio-Rad) for about 30 min.
The gels used for western blotting were transferred onto nitrocellulose membrane. Membranes were blocked with 5% milk in TBS-T (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1% Tween-20) at 4 °C for 1 h or overnight. For His-tagged samples, membranes were incubated with 1:5,000 HRP-conjugated anti-6×His (no. MA1-21315-HRP, Invitrogen) in 1% milk in TBS-T for 1 h. Alternatively, membranes were incubated with 1:1,000 polyclonal rabbit anti-all3324 (anti-Cis1) or anti-all3325 (anti-Cis2) (GenScript) and 1:5,000 secondary HRP-conjugated goat anti-rabbit IgG (Abcam) in 1% milk in TBS-T for 1 h, respectively. Membranes were washed three times for 10 min in TBS-T between and after antibody incubations. Signals were detected using a chemiluminescent substrate (ECL, Amersham).
The gels used for western blotting were transferred onto nitrocellulose membrane. Membranes were blocked with 5% milk in TBS-T (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1% Tween-20) at 4 °C for 1 h or overnight. For His-tagged samples, membranes were incubated with 1:5,000 HRP-conjugated anti-6×His (no. MA1-21315-HRP, Invitrogen) in 1% milk in TBS-T for 1 h. Alternatively, membranes were incubated with 1:1,000 polyclonal rabbit anti-all3324 (anti-Cis1) or anti-all3325 (anti-Cis2) (GenScript) and 1:5,000 secondary HRP-conjugated goat anti-rabbit IgG (Abcam) in 1% milk in TBS-T for 1 h, respectively. Membranes were washed three times for 10 min in TBS-T between and after antibody incubations. Signals were detected using a chemiluminescent substrate (ECL, Amersham).
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