Harvested tumors were paraffin-embedded, processed, and blocked for unspecific antibody staining using rabbit serum (Vectastain ABC HRP kit, Vector Laboratories, Burlingame, CA, USA). Subsequently, tissue sections were stained with the primary antibody goat anti-mouse CREG1 (R&D Systems; AF1697) overnight in a humidified chamber at 4 °C. After rinsing slides in PBS-Tween, they were probed with the secondary antibody anti-goat IgG (Vectastain ABC HRP kit, Vector Laboratories) for 45 min in a humidified chamber at room temperature. Subsequently, ABC complex solution (Vectastain ABC HRP kit, Vector Laboratories) was applied to increase sensitivity for 45 min in a humidified chamber at room temperature. Lastly, a 3,3′-diaminobenzidine (DAB) substrate solution (Sigma-Aldrich) was added and the reaction was stopped by ddH2O. Counterstaining was achieved with Mayer’s hemalum solution (VWR, Radnor, PA, USA). Afterward, slides were dried and mounted with Aquatex (Merck Millipore, Burlington, MA, USA). Tissue sections were imaged using an Axioskop2/AxioCam microscope (Carl Zeiss, Jena, Germany) and analyzed using AxioVision software (Carl Zeiss) and Fiji/ImageJ (NIH, Bethesda, MD, USA).
Vectastain abc hrp kit
Manufactured by Vector Laboratories
Sourced in United States, United Kingdom, Denmark
The Vectastain ABC HRP kit is a detection system used in immunohistochemistry and other immunoassays. The kit contains reagents that amplify the signal from the primary antibody, allowing for improved sensitivity and visualization of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Dab peroxidase hrp substrate kit, by Vector Laboratories (5 mentions)
Dab chromogen substrate, by Merck Group (5 mentions)
3 3 diaminobenzidine (dab), by Merck Group (4 mentions)
Diaminobenzidine dab, by Merck Group (4 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (4 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (4 mentions)
Mayer s hematoxylin, by Merck Group (3 mentions)
Immpact dab peroxidase hrp substrate, by Vector Laboratories (3 mentions)
Mayer s hemalum solution, by Merck Group (3 mentions)
Microscope ch9435 hee56rbrugg, by Leica (3 mentions)
Ab62623, by Abcam (2 mentions)
Ab64715, by Abcam (2 mentions)
Ab28052, by Abcam (2 mentions)
Ab9722, by Abcam (2 mentions)
Laminin, by Abcam (2 mentions)
Image pro plus 7.0, by Adobe (2 mentions)
Immpact dab peroxidase substrate, by Vector Laboratories (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Apoptag peroxidase in situ apoptosis detection kit, by Merck Group (2 mentions)
Dual endogenous enzyme blocking reagent, by Agilent Technologies (2 mentions)
123 protocols using vectastain abc hrp kit
1
Immunohistochemical Staining of CREG1
2
Immunocytochemical and Immunofluorescence Staining of α-SMA
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Cells seeded on Lab-Tek®II Chamber SlideTM system (Thermo Fisher Scientific Inc., Rochester, NY, USA) were fixed with PLP solution for 45 minutes61 (link).
For immunocytochemical staining, endogenous peroxidase was blocked with 3% H2O2 and cells were pre-incubated with serum14 (link), 20 (link). Primary antibody against α-SMA (Abcam) was used. After washing, cells were incubated with secondary antibody (biotinylated IgG; Sigma), washed, and processed using Vectastain ABC-HRP kits (Vector Laboratories Inc.). Cells were counterstained with hematoxylin and mounted with Biomount medium on slides as previously described14 (link), 20 (link).
For immunofluorescence, cells were pre-incubated with serum. Primary antibodies against α-SMA (Abcam) and affinity-purified Alexa Fluor 488-conjugated secondary antibody (Life Technologies Corporation, USA) were used. Nuclei were labeled with DAPI (Sigma-Aldrich, USA) and mounted with medium to prevent quenching (Vector Laboratories Inc., USA)61 (link).
For immunocytochemical staining, endogenous peroxidase was blocked with 3% H2O2 and cells were pre-incubated with serum14 (link), 20 (link). Primary antibody against α-SMA (Abcam) was used. After washing, cells were incubated with secondary antibody (biotinylated IgG; Sigma), washed, and processed using Vectastain ABC-HRP kits (Vector Laboratories Inc.). Cells were counterstained with hematoxylin and mounted with Biomount medium on slides as previously described14 (link), 20 (link).
For immunofluorescence, cells were pre-incubated with serum. Primary antibodies against α-SMA (Abcam) and affinity-purified Alexa Fluor 488-conjugated secondary antibody (Life Technologies Corporation, USA) were used. Nuclei were labeled with DAPI (Sigma-Aldrich, USA) and mounted with medium to prevent quenching (Vector Laboratories Inc., USA)61 (link).
3
Histopathological Analysis of Tumor Tissues
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Isolated tumors and lungs were fixed in 4% paraformaldehyde and paraffin-embedded. Five micrometer sections were deparaffinized and stained by different methods. Tumors were stained by HE (hematoxylin-eosin) solution (Merck Millipore, Darmstadt, Germany), or by TUNEL staining using the ApopTag® Peroxidase in Situ Apoptosis Detection Kit, (#S7100, Sigma-Aldrich, Basel, Switzerland), or by Cleaved-caspase 3 (Asp175) IHC (#9664, CST, Danvers, MA, USA). Anti-Ki67 (#ab15580, Abcam, Cambridge, UK) enabled the detection of metastasis in lung slides. Detection was performed using the Vectastain Elite ABC kit (Vectastain ABC HRP kits, #PK-4010 (mouse)/#PK-4001 (rabbit) Vector Laboratories, Burlingame, CA, USA), followed by 3,3-diaminobenzidine (DAB) (Sigma) incubation prior to mounting. Staining of CD 31+ cells was performed with APC Rat Anti-Mouse CD31 Clone MEC 13.3 (#551262, BD Biosciences, Allschwil, Switzerland). TUNEL stained areas were quantified using the Image J software.
4
Immunohistochemical Analysis of Cellular Markers
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Immunohistochemical staining was performed as previously described. Primary antibodies against p16 (ab211542; Abcam), p53 (sc‐126; Santa Cruz Biotechnology), acetyl‐histone H3 (Lys9/Lys14; #9677; Cell Signaling Technology), 8‐OHdG (ab62623; Abcam), IL‐1β (ab9722; Abcam), CD3e (sc‐20,047; Santa Cruz Biotechnology), IL‐6 (sc‐1265; Santa Cruz Biotechnology), TNF‐α (sc‐52,746; Santa Cruz Biotechnology), α‐SMA (ab28052; Abcam), Collagen 1 (#1310‐08; Southern Biotech), TGF‐β1 (ab64715; Abcam), and IL‐11 (sc‐133,063; Santa Cruz Biotechnology), and IL‐11Rα1 (sc‐130,920; Santa Cruz Biotechnology). After washing, the sections were incubated with secondary antibody (biotinylated IgG; Sigma‐Aldrich), washed, and processed using Vectastain ABC‐HRP kits (Vector Laboratories).
5
Immunohistochemical Staining of Biomarkers
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Staining was performed as previously described4 (link),5 (link),23 (link). Primary antibodies against p53 (#2524, Cell Signaling Technology, Beverly, MA, USA), 8-OHdG (ab62623, Abcam, Cambridge, MA, USA), IL-1β (ab9722, Abcam), IL-6 (sc-1265, Santa Cruz Biotechnology Inc., Dallas, TX, USA), TNF-α (sc-52746, Santa Cruz Biotechnology Inc.), α-SMA (ab28052, Abcam), collagen 1 (#1310-08, Southern Biotech, Birmingham, AL, USA), fibronectin (#SAB4500974, Sigma-Aldrich), TGF-β1 (ab64715, Abcam), and IL-11 (sc-133063, Santa Cruz Biotechnology Inc.). After washing, the sections were incubated with secondary antibody (biotinylated IgG; Sigma-Aldrich), washed and processed using Vectastain ABC-HRP kits (Vector Laboratories Inc., Burlingame, CA, USA).
6
Atherosclerosis Histopathological Characterization
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Anesthetized mice were perfused with 10% formalin in buffered saline for 5 min before dissection of the heart and the entire aorta to the iliac bifurcation. The tissues were stored in 10% buffered formalin solution for 1 week. The aortas were opened longitudinally and stained with Oil Red O for 30 min, whereas the top half of the heart was cryopreserved in 4% paraformaldehyde at 4 °C overnight before imbedding in OCT compound for frozen section preparation. Eight cryosections of 7-μm thickness through the aortic valve region were stained with Oil Red O for 15 min and counterstained with hematoxylin for 3 min. Composition of the atherosclerotic lesions was determined by staining serial sections for 1 h with Sirius Red to identify fibrotic areas and immunohistochemical analysis with antibodies against CD68, IL-1β, or nitrotyrosine. Immunohistochemical analysis was performed using VECTASTAIN ABC-HRP kits (Vector Laboratories) according to manufacturer's instructions. Images were obtained using an Olympus BX6 microscope and digitalized for quantitative analysis using ImageJ software as described (88 (link)).
7
Immunohistochemical Analysis of Breast Cancer
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IHC was performed on a breast cancer tumor microarray (BR1141A; tissuearray.com). Antibodies used for IHC include pHSF1 (S326; Abcam #76076; RRID: AB_1310328), CD3 (Santa Cruz Biotechnology #sc-20047; RRID: AB_627014), CD8A (CST #85336; RRID: AB_2800052), and CCL5 (Invitrogen #710001; RRID: AB_2532515). IHC was performed by deparaffinization using xylenes followed by rehydration with decreasing alcohol solutions. Slides were then incubated in Bloxall (Vector Laboratories #SP-6000–100) and subjected to antigen retrieval with 10 mmol/L citrate solution in a heated pressure cooker (2,100 Antigen Retriever; Aptum Biologics). Tissues were blocked in animal-free blocker (Vector Laboratories #SP-5035) and incubated in primary antibodies overnight in a humidified chamber. Primary antibodies were detected using Vectastain ABC-HRP kits (Rabbit: PK-4001; Mouse: PK-4010) and developed with DAB Immpact kit (Vector Laboratories #SK-4105) followed by dehydration with increasing alcohol solutions and mounted. Slides were imaged with Motic EasyScan scanner and analyzed with QuPath software (27 (link)).
8
Immunohistochemical Quantification of Vascular Markers
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Using VECTASTAIN ABC-HRP Kits (Vector Labs, Burlingame, CA, USA), 10 μm sections were stained with antibodies against CD68 (biotinylated clone FA-11, 1:10, AbD Serotec, Oxford, UK), CD31 (1:100, BD Pharmingen), laminin (1:300, Abcam), α-actin (clone ab5694 1:200, Abcam, Cambridge, UK), or anti-TSP-1 Ab4 (clone 6.1 1:100, Thermo, Waltham, MA, USA). Secondary antibodies were included in the species-specific kit, followed by ImmPACT DAB peroxidase substrate (Vector Labs). Slides were scanned using Leica SCN400 or Aperio AT2 at 20× magnification. Positive staining in the images was quantified using Photoshop CS2 (Adobe, San Jose, CA, USA) and Image Pro Plus (7.0).
9
Immunohistochemical Analysis of PCNA and Collagen I
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Bone sections were air-dried and endogenous peroxidase was blocked with 3% H2O2 and cells were pre-incubated with serum. Primary antibody against PCNA (sc-130920, Santa Cruz Biotechnology Inc., USA) and type-1 collagen (collagen 1) (ab88147, Abcam, USA) was used. After washing, bone sections were incubated with secondary antibody (biotinylated IgG; Sigma), washed, and processed using Vectastain ABC-HRP kits (Vector Laboratories Inc., USA).
10
Immunohistochemical Staining and Quantification
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Using VECTASTAIN ABC-HRP Kits (Vector Labs), 10 µm sections were stained with antibodies against CD68 (biotinylated clone FA-11, 1:10, AbD Serotec), CD31 (1:100, BD Pharmingen), laminin (1:300, Abcam), α-actin (clone ab5694 1:200, Abcam), or anti-TSP-1 Ab4 (clone 6.1 1:100, Thermo). Secondary antibodies were included in the species-specific kit, followed by ImmPACT DAB peroxidase substrate (Vector Labs). Slides were scanned using Leica SCN400 or Aperio AT2 at 20X magnification. Positive staining in the images was quantified using Photoshop CS2 (Adobe) and Image Pro Plus (7.0).
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