AEG-1, HIF-1α, and VEGF protein levels were assessed immunohistochemically in biopsy samples after paraffin-embedding, by the avidin-biotin immunoperoxidase method, as instructed by the manufacturer. After dewaxing and rehydration by standard methods, the sections were incubated with primary antibodies targeting human AEG-1 (ab45338, Abcam), HIF-1α (ab16066, Abcam), and VEGF (ab155944, Abcam) overnight at 4°C, respectively, followed by incubation with biotin-conjugated secondary antibodies (Santa Cruz, USA). Negative control slides were incubated with rabbit serum in lieu of primary antibodies.
Biotin conjugated secondary antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Biotin-conjugated secondary antibody is a laboratory reagent used to detect and visualize target proteins in various immunoassays. It consists of a secondary antibody that has been conjugated with the molecule biotin. The biotin moiety allows for the subsequent detection and amplification of the signal, typically through the use of a streptavidin-based detection system.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Pall Corporation (2 mentions)
Ab16066, by Abcam (1 mentions)
Ab45338, by Abcam (1 mentions)
Ab155944, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Bio-Rad (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Protein blocking reagent, by Agilent Technologies (1 mentions)
Ab151562, by Abcam (1 mentions)
Ab64957, by Abcam (1 mentions)
Tnf α, by Santa Cruz Biotechnology (1 mentions)
Ifn γ, by Santa Cruz Biotechnology (1 mentions)
Tgf β, by Santa Cruz Biotechnology (1 mentions)
Antigen unmasking solution, by Vector Laboratories (1 mentions)
Dab peroxidase kit, by Vector Laboratories (1 mentions)
Hematoxylin, by Vector Laboratories (1 mentions)
Axioskop 2 plus fluorescence microscope, by Zeiss (1 mentions)
Alexa fluor 488 streptavidin, by Jackson ImmunoResearch (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Hoechst 33342, by AAT Bioquest (1 mentions)
Pparγ, by Abcam (1 mentions)
13 protocols using biotin conjugated secondary antibody
1
Immunohistochemical Analysis of AEG-1, HIF-1α, and VEGF
2
Cell Cycle Regulation in Murine Liver Samples
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On days 21 and 42 of the experiment, liver samples were collected from eight mice per group and the expression of cell cycle regulatory proteins was examined by western blot. The liver was homogenized and proteins were extracted with RIPA lysis buffer and kept in Laemmli loading buffer. Protein samples were resolved on SDS-PAGE gels (5%–15%) and transferred to nitrocellulose filter membranes. Membranes were blocked with 5% fat-free milk for 1h and incubated with primary antibodies overnight at 4°C. The primary antibodies were cyclin D/E/B/A, CDK1/2/4, p-ATM, p-p53, p21, p-ATR, p-Chk1, and p-Cdc25A (Table 1 ). The membranes were then washed with PBS-Tween, incubated with biotin-conjugated secondary antibodies (Santa Cruz, USA) for 1h, and washed again with PBS-Tween. Blots were visualized by ECL™ (Bio-Rad, Hercules, CA, USA) and X-ray film. Statistical analysis of protein expression was performed using ImageJ2x software.
3
Immunohistochemical Analysis of Prostate
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At least three 4-micrometer sections were taken from different mouse prostates for each of the genotypes surveyed. Sections were used for H&E staining and for PRR and PACE IHC. Slides were heated for 20 minutes at 60˚C, deparaffinized in xylenes and rehydrated in ethanol gradient. Antigen retrieval was performed using EDTA pH 8.0 (PRR) buffer and sodium citrate pH 6.0 (PACE4). PACE4 slides were further autoclaved in 10 mM citrate buffer pH 6 for 45 min (16 psi, 121 ˚C). To deactivate endogenous peroxidase, 3% H2O2 was used, and slides were blocked using a protein blocking reagent (Dako) for PRR and an IHC blocking buffer (Pierce) for PACE4, at room temperature. Tissue sections were incubated with primary antibodies diluted in 5% BSA in TBST overnight at 4˚C (Abcam, ab64957, 1:600; PACE4: Abcam, ab151562, 1:400) and then with biotin-conjugated secondary antibodies (Santa-Cruz) for PRR and a secondary HRP-conjugated anti-Rabbit antibody (Biorad) for PACE4. Staining was developed using DAB (Sigma-Aldrich) containing 0.015% H2O2. Counterstaining was performed with hematoxylin.
4
Cytokine Expression in Murine Spleen
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At 21 and 42 days of age, splenic samples of eight mice in each group were taken to determine the cytokine protein expression levels by western blot.
The spleen was ground into homogenate and then proteins were extracted with RIPA lysis buffer and kept in laemmli loading buffer. Protein samples were resolved on SDS-PAGE (10%–15% gels) and transferred to nitrocellulose filter membranes. Membranes were blocked with 5% fat-free milk for 1h and incubated with primary antibodies overnight at 4°C. The primary antibodies were TNF-α, IFN-γ, TGF-β, and IL-2, IL-10 (Santa Cruz, USA). The membranes were then washed with PBS-tween and incubated with biotin-conjugated secondary antibodies (Santa cruz, USA) for 1h, and washed again with PBS-tween. Blots were visualized by ECLTM (Bio-Rad, Hercules, CA, USA) and X-ray film.
The spleen was ground into homogenate and then proteins were extracted with RIPA lysis buffer and kept in laemmli loading buffer. Protein samples were resolved on SDS-PAGE (10%–15% gels) and transferred to nitrocellulose filter membranes. Membranes were blocked with 5% fat-free milk for 1h and incubated with primary antibodies overnight at 4°C. The primary antibodies were TNF-α, IFN-γ, TGF-β, and IL-2, IL-10 (Santa Cruz, USA). The membranes were then washed with PBS-tween and incubated with biotin-conjugated secondary antibodies (Santa cruz, USA) for 1h, and washed again with PBS-tween. Blots were visualized by ECLTM (Bio-Rad, Hercules, CA, USA) and X-ray film.
5
Intestinal Immunohistochemistry for ARG1 and NOS2
6
Protein Expression Profiling in Cells
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Cells were lysed, and proteins were extracted with RIPA lysis buffer and kept in Laemmli buffer. Protein samples were resolved on SDS-PAGE (10%–15% gels) and transferred to nitrocellulose filter membranes. Membranes were blocked with 5% fat-free milk for 1 h and incubated with primary antibodies overnight at 4°C. The primary antibodies were cyclin D/E/B/A, CDK1/2/4 (Abcam, UK) and TNF-α, IFN-γ, TGF-β, and IL-2, IL-10 (Santa Cruz, USA). The membranes were then washed with PBS-tween and incubated with biotin-conjugated secondary antibodies (Santa cruz, USA) for 1 h, and washed again with PBS-tween. Blots were visualized by ECLTM (Bio-Rad, Hercules, CA, USA) and X-ray film.
7
Protein Expression Analysis of PA Treatment
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After culturing in a 100 mm dish, 2 × 106 cells were incubated with IC60 concentration of PA (112 μM in A549; 135 μM in A549/V16) for 0, 6, 12, 24, and 48 h. Total protein was extracted from the cells using RIPA buffer (Visual Protein) supplemented with protease (Amresco Inc.) and phosphatase inhibitors (Bionovas) on ice for 30 min. After quantification of proteins using the BCA protein assay kit (Pierce; Thermo Scientific), the equivalent amount of protein (20 μg) was separated using 8%–12.5% SDS‐PAGE gel and transferred to PVDF membrane (PALL Corporation), followed by blocking with 5% skim milk. Membranes were washed with 0.5% tween‐20 in TBS (TBS‐T) and immunolabeled with primary antibodies at 4°C overnight. Membranes were washed thrice with TBS‐T and then incubated with biotin‐conjugated secondary antibody (Santa Cruz) for 2 h at 25°C, followed by peroxidase‐conjugated streptavidin incubation (Jackson ImmunoResearch Inc.) for 1 h at 25°C. The bands were visualized using an enhanced chemiluminescence reagent (ECL, T‐Pro Biotechnology) and a GE LAS‐4000 chemiluminescence imaging analyzer (GE Healthcare Life Sciences). Protein blots were quantified by densitometry using ImageJ software (National Institute of Health), and the amounts were expressed relative to the internal reference β‐actin.
8
Quantifying Cancer Stem Cell Markers
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A549 and A549/V16 cells (1.5 × 105 cells/well) were plated onto 15 mm coverslips (Assistant) in a 35‐mm dish and exposed to IC60 concentration of PA for 48 h. The cells were fixed with 10% neutral buffered formalin, permeabilized with 1% NP‐40 in PBS, blocked with 5% bovine serum albumin (Sigma‐Aldrich), and then incubated with antibody against P‐gp, CD133, CD44, and EpCAM overnight at 4°C. The cells were incubated with a biotin‐conjugated secondary antibody (Santa Cruz, CA, USA) for 2 h, followed by incubation with Alexa Fluor 488 streptavidin (Jackson ImmunoResearch Inc.) for 1 h. Nuclei were stained with Hoechst 33342 (AAT Bioquest), and images were acquired using a Zeiss Axioskop 2 plus fluorescence microscope (Carl Zeiss).
9
Quantifying Placental Protein Expression
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The total protein content of placenta extracts was determined by Bradford’s method [28 ]. Ten μg of total protein were resolved in 10% or 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes after electrophoresis. Protein ladders were run in parallel to estimate molecular weights. Membranes were blocked with 5% bovine serum albumin in PBST (Phosphate Buffered Saline, 0.1% Tween 20) and incubated overnight at 4 °C with primary antibodies against PPARγ (1:2000; Abcam, Cambridge, MA, USA), FATP1 (1:1000; Abcam, Cambridge, MA, USA), FATP4 (1:1000; Abcam, Cambridge, MA, USA) and actin (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were washed, incubated for 1 h with the appropriate biotin-conjugated secondary antibody (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and then incubated with horseradish peroxidase-conjugated streptavidin (1:1000). Immunoreactive proteins were visualized using the ECL system. Films were scanned and bands were quantified by densitometry with ImageJ 1.34 s Software (NIH, Bethesda, MD, USA), using actin as internal control.
10
Immunohistochemical Analysis of p-Akt in Femoral Heads
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Femoral heads in each group were harvested and fixed with 4% paraformaldehyde for 1 day and processed for paraffin embedding. Following embedding in paraffin, samples were cut into 5-μm sections. Subsequently, the sections were dewaxed and hydrated, followed by HE staining according to the instruction manual. Following deparaffinization, epitope retrieval was performed in a citrate buffer (pH 6.0) heated in a microwave oven for 10 min. The sections were incubated with 20% BSA blocking solution (Solarbio, Beijing, China, 37 °C, 30 min) for blocking non-specific staining. The slides were then incubated with p-Akt primary antibody (Cell Signaling, Cat#4060S, RRID: AB_2315049) at 4 °C overnight, followed by incubation with biotin-conjugated secondary antibody (1:1000; sc-2004; Santa Cruz Biotechnology, Inc.). Photographs were taken with an Olympus Optical AX70 microscope (Olympus).
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