Five-micrometer sections of formalin-fixed and paraffin-embedded tumor tissue were mounted on charged glass slides and dried at 58 °C for 60 min. Slides were first deparaffinized with xylene then epitope retrieval carried out using citrate buffer in a 2100 Retriever pressure cooker (PickCell Laboratories, San Jose, CA, USA). The sections were blocked with 10% goat serum for 1 h, then incubated with primary antibody (anti-p27, 1 : 50, Abcam (ab7961)) overnight, followed by an HRP-linked goat anti-rabbit secondary antibody (1 : 300, Pierce Biotechnology). The sections were developed with 3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories, Burlingame, CA, USA) for 2 min and nuclei counterstained with hematoxylin (Sigma-Aldrich, St. Louis, MO, USA) for 2 min. PBS washes were performed between all steps. The slides were dehydrated in graded alcohols (100%, 95% and 80% ethanol (vol/vol) in H2O), cleared in xylene and coverslips attached with Permount (Fisher Scientific, Waltham, MA, USA). Slides were scanned using a ScanScope XT Digital Slide Scanner (Aperio, Vista, CA, USA) and data were analyzed with the positive pixel count algorithm (ImageScope, Aperio).
3 3 diaminobenzidine tetrahydrochloride
Manufactured by Vector Laboratories
Sourced in United States
3,3′-diaminobenzidine tetrahydrochloride is a chromogenic substrate used in immunohistochemistry and enzyme-linked immunosorbent assays (ELISA) to detect the presence of target analytes. It produces a brown-colored reaction product when oxidized by peroxidase enzymes.
Automatically generated - may contain errors
Lab products found in correlation
Target retrieval solution, by Agilent Technologies (4 mentions)
Vectastain abc kit, by Vector Laboratories (4 mentions)
Envision system hrp, by Agilent Technologies (3 mentions)
Biotinylated secondary antibody, by Vector Laboratories (3 mentions)
Vectastain abc, by Vector Laboratories (3 mentions)
Avidin biotin complex, by Vector Laboratories (3 mentions)
Tsa plus dig kit, by PerkinElmer (2 mentions)
Tween 20, by Merck Group (2 mentions)
Mayer s hematoxylin, by Merck Group (2 mentions)
Permount, by Merck Group (2 mentions)
Blocking serum from abc vectastain kit, by Vector Laboratories (2 mentions)
Anti myc, by Abcam (2 mentions)
Hematoxylin, by Merck Group (1 mentions)
Permount, by Thermo Fisher Scientific (1 mentions)
Ab7961, by Abcam (1 mentions)
Hrp linked goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Quantigene viewrna ish tissue assay kit, by Thermo Fisher Scientific (1 mentions)
Ab9574, by Abcam (1 mentions)
Blocking agent, by Thermo Fisher Scientific (1 mentions)
Cd11c ep1347y, by Abcam (1 mentions)
31 protocols using 3 3 diaminobenzidine tetrahydrochloride
1
Immunohistochemical Quantification of p27 in FFPE Tumors
2
Immunohistochemical Analysis of Liver Markers
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The livers were fixed in phosphate-buffered 10% formalin for 24 h, and paraffin sections were then prepared. Immunohistochemical staining was carried out with the EnVision/HRP system (Dako, Carpinteria, CA, USA) on deparaffinized sections treated with Target Retrieval Solution (Dako). The antibodies used were as follows: anti-insulin-like growth factor 2 (IGF2) (ab9574; Abcam, Cambridge, UK), anti-α-fetoprotein (AFP) (14550-1-AP, for mouse tissues; Proteintech Group, Chicago, IL, USA), anti-AFP (A0008, for human tissues; Dako), and anti-trefoil factor 3 (TFF3) (Abbiotec, San Diego, CA, USA). 3,3′-Diaminobenzidine tetrahydrochloride (Vector Laboratories, Burlingame, CA, USA) was used for signal detection. For the detection of the TFF3 peptide, we applied signal amplification using the TSA Plus DIG Kit (PerkinElmer, Waltham, MA, USA). In situ hybridization for non-coding H19 mRNA was carried out on deparaffinized sections using the mouse H19 QuantiGene ViewRNA Probe Set (VB6-16706; Affymetrix, Santa Clara, CA, USA) and the QuantiGene ViewRNA ISH Tissue Assay Kit (Affymetrix).
3
Immunohistochemical Analysis of LDHB and PDH
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Tumor tissues were fixed with 10% neutral-buffered formalin for 24 h and embedded in paraffin. Paraffin-embedded 5-μm sections were deparaffinized and rehydrated, washed in distilled water, and subjected to heat-mediated antigen retrieval. Endogenous peroxidase activity was quenched by treatment with 3% H2O2 for 30 min, and sections were washed with phosphate-buffered saline for 5 min. The sections were blocked for 1 h with a blocking agent (Invitrogen) and incubated overnight with antibodies against LDHB or PDH (1:1000, Cell Signaling) at 4 °C. The sections were then incubated at room temperature with a biotinylated secondary antibody (1:500, Vector Laboratories, Burlingame, CA, USA) for 1 h. The slides were washed and treated with 3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories). Sections were then counterstained with hematoxylin and mounted in aqua mount (Vector Laboratories).
4
Immunohistochemical Analysis of CINC-1 in Lung
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For the immunohistochemistry analysis, the lung sections were subjected to a high temperature (121°C) for 1 minute for antigen retrieval. After blocking of nonspecific sites with 3% hydrogen peroxide for 5 minutes, the specimens were stained with rabbit polyclonal CINC-1 antibody (Millipore; 1:100 dilution, Temecula, California, USA). The reactions were stained with Vectastain ABC (Vector Laboratories). The colour was developed with 3,3-diaminobenzidinetetrahydrochloride (Vector Laboratories) and counterstained with H&E.
5
Liver Injury and Inflammation Analysis
6
Immunohistochemical Analysis of Placental Immune Cells
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Paraffin-embedded full thickness placental samples were sliced into 3 µm sections, dewaxed and heated in 0.01 M pH 6 sodium citrate buffer for antigen retrieval. After blocking endogenous peroxidase activity and non-specific binding, the sections were incubated overnight with the following primary antihuman antibodies: mouse monoclonal anti-CD3 (F7.2.38, 1:70, Dako, Glostrup, Denmark), mouse monoclonal anti-CD20 (L26, 1:126, Dako), mouse monoclonal anti-CD68 (PG-M1, 1:30, Dako), rabbit monoclonal anti-CD11c (EP1347Y, 1:500, Abcam, Cambridge, UK) and rabbit polyclonal anti-ACKR2 (1:400, Sigma-Aldrich, St Louis, MO, USA). Sections were then incubated with the appropriate chromogenic secondary antibody (ImmPRESS Polymer Detection Kit, anti-rabbit and anti-mouse, Vector Laboratories, Burlingame, CA, USA). The immunoreactivity was developed using 3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories) as chromogen. Isotype-matched control antibodies were included as a negative control and tonsil sections as a positive control. The sections were observed under a light microscope (Olympus BX43, Olympus, Tokyo, Japan) and photographed by digital camera (DP22 using Olympus Cell Sense Entry 2.2 for imaging acquisition).
7
Quantifying Macrophage Subsets via Immunohistochemistry
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Sections were deparaffinized and hydrated and the slides were incubated with 10 mM sodium citrate. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Slides were washed in TBS with 0.05% Tween-20 (Sigma), blocked with serum-free protein block (Dako, Carpinteria, CA, USA), and immunostained with Vectastain ABC (Vector Laboratories, Inc., Burlingame, CA, USA). Antibodies against CD68 (1:100 dilution, 125212; Abcam), CD163 (1:100 dilution, 87099; Abcam), and NOS 2 (1:350 dilution, N-20; Santa Cruz Biotechnology) were incubated in TBS/Tween buffer overnight at 4 °C. Color was developed with 3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories, Inc.) and counterstained with H&E. An isotype immunoglobulin G (IgG) was used as negative control. CD68+ and CD163+ macrophage densities were assessed in five fields of view in the alveolar space by the point-counting technique, using a 100-point grid of known area (104 μm2) attached to the eyepiece of the microscope [33 ]. The point-counting technique was assessed independently by one coauthor (VM), who was blinded to group allocation.
8
Immunohistochemical Analysis of Prostate Cancer
9
Immunohistochemistry Protocol for Coronal Sections
10
Histological Analysis of Carotid Arteries
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ApoE-/- mice were perfused with vasodilation buffer as described earlier. Dissected carotid arteries were stored overnight in 4% PFA at 4 °C and embedded in paraffin. 4 µm sections were counterstained with hematoxylin and eosin (H/E). In addition, 4 µm sections were incubated with target retrieval solution pH9 (Agilent, Santa Clara, CA, United States) and with antisera raised against murine CD31 (ThermoFisher Scientific, Dreieich, Germany). The slides were subsequently incubated with secondary antibody and stained using 3,3-diaminobenzidine-tetra-hydrochloride as chromogenic substrate (Vector Laboratories, Peterborough, United Kingdom). Sections were counterstained with hematoxylin and mounted.
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