OCT-embedded, frozen tumor tissues were cryosectioned in a Leica CM3050 S Cryostat at 10μm thickness for all immunohistochemical (IHC) and immunofluorescence (IF) studies. Slides were incubated overnight in primary antibody at 4°C. Secondary/tertiary antibodies were added and sections were developed and counterstained according to the appropriate protocol. For IHC, the following antibodies were used: anti-human fibronectin (Sigma #F3648), anti-mouse versican (EMD Millipore #AB1033, Burlington, MA, USA), biotinylated hyaluronic acid binding protein, HABP (EMD Millipore #385911), Rabbit IgG isotype control (Santa Cruz Biotechnology #sc-2027, Dallas, TX, USA), and biotin-SP-conjugated goat anti-rabbit IgG secondary antibody (Jackson Immunoresearch #AB2337959, West Grove, PA, USA). The following antibodies were used for IF: anti-human FAP antibody (R&D Systems #AF3715, Minneapolis, MN, USA or Abcam #53066, Cambridge, MA, USA) or sheep IgG isotype control (R&D Systems #5001A), and AF488-conjugated donkey anti-sheep IgG secondary antibody (ThermoFisher #A11015, Waltham, MA, USA). All imaging was completed with the Olympus Spot-RT3 microscope at 20X magnification. For quantification purposes, 3-5 different tumor samples (n≥3) from each experimental group were used. The mean area of positive staining and mean fluorescence intensity were determined using Fiji ImageJ Software.
Rabbit igg isotype control
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Rabbit IgG Isotype Control is a laboratory reagent used as a control in immunoassays. It consists of purified rabbit immunoglobulin G (IgG) that serves as a reference for evaluating the specific binding of antibodies in experimental samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab1033, by Merck Group (2 mentions)
Anti mouse versican, by Merck Group (2 mentions)
Biotin sp conjugated goat anti rabbit igg secondary antibody, by Jackson ImmunoResearch (2 mentions)
Af488 conjugated donkey anti sheep igg secondary antibody, by Thermo Fisher Scientific (2 mentions)
Spot rt3 microscope, by Olympus (2 mentions)
Anti human fibronectin, by Merck Group (2 mentions)
Af3715, by Abcam (2 mentions)
Cm3050s cryostat, by Leica camera (2 mentions)
Axiocam digital camera, by Zeiss (2 mentions)
Tigar, by Abcam (1 mentions)
Mouse igg isotype control, by Santa Cruz Biotechnology (1 mentions)
Anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Dmi6000b inverted microscope, by Leica camera (1 mentions)
3 amino 9 ethylcarbazole aec, by Vector Laboratories (1 mentions)
Axioplan 2 microscope, by Zeiss (1 mentions)
Ez chip chromatin immunoprecipitation kit, by Merck Group (1 mentions)
7 protocols using rabbit igg isotype control
1
Immunohistochemical Analysis of Tumor Tissues
2
Immunodetection of Cell Signaling Proteins
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Bax 1:1000 (AbCam), Bak1 1:1000 (AbCam), p21 1:1000 (Cell signaling), TP53 D01 1:200 (Santa Cruz), TP53 D01 ChIP Grade 1 μg/ul (AbCam), TP53 D07 1:100 (Millipore), TP73 1:200 (Santa Cruz), DAPI (10 nM) (Invitrogen), G6PD 1:1000 (AbCam), TIGAR 1:200 (AbCam), β-Actin 1:1000 (Santa Cruz), Anti-Mouse IgG HRP 1:5000 (Santa Cruz), Anti-Rabbit IgG HRP 1:5000 (Santa Cruz), Anti-mouse AF633 5 µg/ml (Invitrogen), Mouse IgG Isotype control 1:1000 (Santa Cruz), Rabbit IgG Isotype control 1:1000 (Santa Cruz), Mouse IgG2a ChIP Grade 1 μg/ul (AbCam).
3
Immunohistochemical Analysis of Tumor Tissues
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OCT-embedded, frozen tumor tissues were cryosectioned in a Leica CM3050 S Cryostat at 10μm thickness for all immunohistochemical (IHC) and immunofluorescence (IF) studies. Slides were incubated overnight in primary antibody at 4°C. Secondary/tertiary antibodies were added and sections were developed and counterstained according to the appropriate protocol. For IHC, the following antibodies were used: anti-human fibronectin (Sigma #F3648), anti-mouse versican (EMD Millipore #AB1033, Burlington, MA, USA), biotinylated hyaluronic acid binding protein, HABP (EMD Millipore #385911), Rabbit IgG isotype control (Santa Cruz Biotechnology #sc-2027, Dallas, TX, USA), and biotin-SP-conjugated goat anti-rabbit IgG secondary antibody (Jackson Immunoresearch #AB2337959, West Grove, PA, USA). The following antibodies were used for IF: anti-human FAP antibody (R&D Systems #AF3715, Minneapolis, MN, USA or Abcam #53066, Cambridge, MA, USA) or sheep IgG isotype control (R&D Systems #5001A), and AF488-conjugated donkey anti-sheep IgG secondary antibody (ThermoFisher #A11015, Waltham, MA, USA). All imaging was completed with the Olympus Spot-RT3 microscope at 20X magnification. For quantification purposes, 3-5 different tumor samples (n≥3) from each experimental group were used. The mean area of positive staining and mean fluorescence intensity were determined using Fiji ImageJ Software.
4
Histological Analysis of Mouse Bladder and Prostate
5
Immunohistochemical Analysis of NPM in Psoriasis
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Paraffin-embedded sections were obtained from biopsies of healthy or psoriatic skin, including LS and NLS areas of evolving plaques. Five-micrometer sections were dewaxed and rehydrated. After quenching endogenous peroxidase, achieving antigen retrieval and blocking non-specific binding sites, sections were incubated with the anti-human NPM rabbit polyclonal antibody (ab15440; 1:200). Rabbit IgG isotype control (Santa Cruz) was used at the same concentration of primary antibody. Secondary biotinylated polyclonal Abs and staining kits were obtained from Vector Laboratories. Immunoreactivity was visualized with peroxidase reaction using 3-amino-9-ethylcarbazole (AEC, Vector Laboratories, Burlingame, CA) in H2O2 and specimen counterstained with hematoxylin. As a negative control, primary Abs was omitted. Stained sections were analyzed with the AxioCam digital camera coupled to the Axioplan 2 microscope (Carl Zeiss AG, Oberkochen, Germany). NPM staining intensity was evaluated by quantitative analysis (Image J color deconvolution) in three adjacent fields of each section by two independent observers, blinded to the status of the specimens. The percentage of IHC positivity areas was shown in dot plots graphs.
6
Chromatin Immunoprecipitation of Estrogen Receptor Alpha
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E2-deprived MCF7 cells (1 × 107 cells per 10 cm plate) were treated for 24 h with DMSO alone, DMSO + E2 or compound + E2. One percent formaldehyde was added for 10 min at room temperature to perform DNA-protein crosslinking and quenched by treatment with 125 mM glycine for 5 min. Cell lysates sonicated with Sonic Dismembrator 550 instrument (Thermo Fisher Scientific, Waltham, MA, USA) to yield DNA fragments of 200–1000 bp in size. Lysates (from 3.3 × 106 cells) were immunoprecipitated with 5 g of anti-ERα antibody or 1 g of rabbit isotype control IgG (Santa Cruz Biotechnology, Dallas, TX, USA) using the EZ-ChIP chromatin immunoprecipitation kit (Millipore, Billerica, MA, USA). Bound DNA was quantified by qPCR (SYBR Green master mix, Invitrogen, Carlsbad, CA, USA) using the following primer sets: pS2 enhancer, forward 5′-GTACACGGAGGCCCAGACAGA-3′ and reverse 5′-AGGGCGTGACACCAGGAAA-3′; GAPDH promoter, forward 5′-TACTAGCGGTTTTACGGGCG-3′ and reverse 5′-TCGAACAGGAGCAGA GAGCGA-3′. The qPCR results are presented as fold enrichment of PCR amplification over control IgG antibody and normalized based on the total input (no precipitated chromatin). Primers for the GAPDH promoter were used as a negative control.
7
ChIP Assay of TGFβ1-Induced Epigenetic Regulation
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ChIP assays were carried out from cross-linked BEAS-2B cells with or without TGFβ1 treatment. Briefly, cells were cross-linked in 1% formaldehyde for 10 min, then glycine was added to quench the formaldehyde at a final concentration of 125 mM. After cell lysis and sonication, cell lysate was incubated overnight with various antibodies: anti-H3K27Ac (Abcam, ab4729), anti-SMAD3 (Abcam, ab28379) or rabbit isotype control IgG (Santa Cruz Biotechnology, sc-2027). Immunoprecipitated chromatin was then collected after incubation with protein A magnetic beads (Pierce, 88,845) at 4 °C for 4 h. After reverse cross-linking, DNA products were purified by MinElute PCR Purification Kit (QIAGEN, 28,006). The Quantitative real-time PCR (qPCR) was performed to identify the relative amount of ChIP products compared to the input by primers targeting human HHIP promoter and enhancer regions. Primer sequences used for ChIP-qPCR are listed in Supplementary Table S2 .
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