Tissues were fixed in 10% formalin, paraffin-embedded, and sections at 4 mm interval were cut from each tissue, and stained with hematoxylin and eosin (H&E) or via immunohistochemistry (IHC). For immunohistochemistry, slides were deparaffinized by xylenes and rehydrated by ethanol gradient, then heated in a pressure cooker using DAKO retrieval buffer (DAKO, Santa Clara, CA). Endogenous peroxidases were quenched in 3% hydrogen peroxide in methanol for 30 min. Tissues were blocked with 0.5% BSA in PBS for 30 min and incubated with primary antibodies against: P16, CD45, CD3, CD68, or Neutrophil Elastase (abcam, Cambridge, MA) 1:100–1:200 overnight at 4 °C. Slides were developed using HRP-conjugated secondary antibodies followed by DAB substrate/buffer (DAKO, Santa Clara, CA).
Dab substrate buffer
Manufactured by Agilent Technologies
Sourced in Denmark, China
DAB substrate buffer is a pre-mixed solution used in immunohistochemistry and immunocytochemistry applications. It provides the necessary components for the detection of target proteins or antigens using a diaminobenzidine (DAB) chromogenic reaction.
Automatically generated - may contain errors
Lab products found in correlation
Dako retrieval buffer, by Agilent Technologies (3 mentions)
Neutrophil elastase, by Abcam (1 mentions)
Tunel apoptosis detection kit, by Yeasen (1 mentions)
Gb111499, by Wuhan Servicebio Technology (1 mentions)
Eclipse c1 ortho fluorescent microscopy, by Nikon (1 mentions)
Celecoxib, by Selleck Chemicals (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Hematoxylin, by Agilent Technologies (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Cell conditioning solution, by Roche (1 mentions)
Dab chromogen, by Agilent Technologies (1 mentions)
Vulcan fast red chromogen kit 2, by Biocare Medical (1 mentions)
Mach 2 double stain 2, by Biocare Medical (1 mentions)
Pe conjugated mouse igg1, by BD (1 mentions)
Rabbit igg, by Agilent Technologies (1 mentions)
Envision dual link system hrp, by Agilent Technologies (1 mentions)
Blockaid blocking solution, by Thermo Fisher Scientific (1 mentions)
Protein block, by Agilent Technologies (1 mentions)
Pertex, by Leica Microsystems (1 mentions)
Axioobserver epifluorescence microscope, by Zeiss (1 mentions)
10 protocols using dab substrate buffer
1
Immunohistochemical Analysis of Tissue Samples
2
Immunohistochemical Analysis of Tumor Proliferation and Apoptosis
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The paraffin-embedded tumor specimens were sectioned into 4 μm slides. To evaluate the expression of Ki-67, slides were blocked and incubated with the antibody targeting Ki-67 (Servicebio, GB111499, 1:300) at 4°C overnight. The next day, slides were incubated with the corresponding second antibody at room temperature for 50 min. Finally, slides were visualized with DAB substrate buffer (DAKO, K5007) and photographed using a light microscope at a magnification of x 400. In addition, the TUNEL apoptosis detection kit (YEASEN, 40306ES20) was used to measure the extent of apoptosis in tumors according to the manufacturer’s instructions. Ortho-Fluorescent Microscopy (Nikon Eclipse C1, Japan) was used to observe the nuclear expression of TUNEL-positive cells at a magnification of x200. Both the ki-67 positive rate and the apoptosis rate were calculated by counting the number of positive cells/total cells in five fields randomly selected with ImageJ software.
3
Immunohistochemistry of PD-L1 and COX-2 in Cells
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COX-2-specific mouse monoclonal antibody (mAb) (clone CX-294), mouse IgG2a, rabbit IgG, the peroxidase blocking reagent goat anti-rabbit + horseradish peroxidase (HRP) visualization reagent, DAB substrate buffer, DAB chromogen, bond wash solution, hematoxylin and EnVision FLEX + rabbit linker kitwere purchased from DAKO. PD-L1-specific rabbit mAb (clone SP-142) was purchased from Spring Bioscience. Cell Conditioning Solution was purchased from Ventana medical Systems. Protein serum block, steady plus 3,3′-diaminobenzidine (DAB) kits, goat anti-mouse IgG dylight 488 and goat anti-rabbit IgG dylight 594 were purchased from Abcam. Bond primary antibody diluents and 4′,6′-diamidino-2-phenylindole (DAPI) were purchased from Leica biosystems. MACH 2 DOUBLE STAIN 2 and vulcan fast red chromogen Kit 2 were purchased from Biocare. The COX-2 inhibitor, celecoxib was purchased from Selleck Chemicals LLC. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) and trypan blue were purchased from Sigma. GAPDH-, Bcl-2- and β-actin specific mAbs were purchased from Cell Signaling Technology. R-phycoerythrin(PE)-conjugated PD-L1-specific mouse mAb [clone MIH1 (RUO)] and PE-conjugated mouse IgG1 were purchased from BD Biosciences.
4
Immunohistochemical Staining Protocol for Paraffin-Embedded Tissues
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Tissues were fixed in formalin and then embedded in paraffin. Tissue slices (4 μm thick) were deparaffinized in xylene and hydrated in ethanol. Antigens were retrieved by boiling in 10 mM sodium citrate buffer (pH 6.0) for 1 h. Endogenous peroxidase was inactivated in 3% H2O2-containing methanol at room temperature for 5 min. Slices were blocked with BlockAid Blocking Solution (Thermo Fisher Scientific) at room temperature for 30 min and were then reacted with primary antibodies (optimally diluted with BlockAid Blocking Solution) at 4 °C overnight. The slides were then washed in PBS and reacted with EnVision+ Dual Link System-HRP (Dako) at room temperature for 30 min. After the slides were washed with PBS, they were stained using DAB Substrate Buffer (Dako) and counterstained with Mayer’s hematoxylin. They were then dehydrated in ethanol and xylene and mounted with coverslips. All images were acquired using an upright microscope (BX63; Olympus, Tokyo, Japan).
5
Immunohistochemical Profiling of Tumor Markers
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FFPE tissue sections at four µm thickness were sliced and stained for integrin αvβ6, CEACAM5, mesothelin, PSMA, uPAR, FAP, ITGA5 and EGFR. After deparaffinization in xylene and rehydration in a stepwise series of alcohol solutions, endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in water for 20 min. Antigen retrieval was performed as described in Supplementary Table 1 . Following antigen retrieval, slides stained for FAP were incubated for 10 min with Protein Block (Dako, Glostrup, Denmark). All slides were incubated overnight at room temperature with primary antibodies (Supplementary Table 1 ). Slides were washed in phosphate-buffered saline (PBS) and incubated for 30 min at room temperature with an HRP-labelled secondary antibody (anti-mouse, anti-rabbit (Envision, Dako, Glostrup, Denmark) or anti-donkey (Invitrogen, Carlsbad, USA)). After being rinsed with PBS, immunoreactions were visualized using DAB substrate buffer (Dako, Glostrup, Denmark) for ten minutes and counterstained using Mayer’s hematoxylin for 30 s. After dehydration at 37 °C, the slides were mounted with PERTEX® (Leica Microsystems, Wetzlar, Germany).
6
Histological Analysis of Rat Pancreas
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After continuous diet treatment for 28 days, the pancreas of the rat was taken and the surface blood was washed with physiological saline, fixed in 10% PBS-paraformaldehyde at 4°C for 24 hours. After paraffin embedding and sectioning (5 μm), tissues were stained with hematoxylin-eosin (H-E) and insulin expression in pancreatic tissue was observed with immunohistochemistry. In detail, paraffin-embedded sections were incubated with the primary anti-insulin antibody (1 : 200; host species : mouse; Boster, China) for 24 h at 4°C and subsequently decorated with the secondary antibody (anti-mouse antibody (1 : 200); Boster, China) for 1 h at room temperature (RT); then, reactions were developed using DAB (3,3′-diaminobenzidine) chromogen and DAB substrate buffer (Dako). All images were acquired with a Zeiss Axio Observer epifluorescence microscope.
7
Prostate Cancer Immunohistochemistry
8
Lung Cancer Tissue Analysis Protocol
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Lung cancer and adjacent normal tissue microarrays were purchased (Biomax, Derwood, MD, Cat #BC04119b, US Biomax, RRID:SCR_004295) and subjected to pathologic examination. Tissues were stained with hematoxylin and eosin (H&E) (Sigma Aldrich), or via immunohistochemistry (IHC). For immunohistochemistry, slides were deparaffinized by xylenes and rehydrated by ethanol gradient, then heated in a pressure cooker using DAKO retrieval buffer (DAKO, Santa Clara, CA). Endogenous peroxidases were quenched in 3% hydrogen peroxide in methanol for 30 minutes. Tissues were blocked with 0.5% BSA in PBS for 30 minutes and incubated with primary antibodies against HAI-1 (abcam, ab189511) or CCR7 (abcam, ab227768) at 1:50–1:200 overnight at 4°C. Slides were developed using HRP conjugated secondary antibodies followed by DAB substrate/buffer (DAKO). All human tissues were from commercially available cell lines and tumor microarrays and not subject to local IRB approval.
9
Tissue Processing and Immunostaining Protocol
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Tissues were fixed in 10% formalin, paraffin-embedded, and sections at 4μm interval were cut from each tissue, and stained with hematoxylin and eosin (H&E), or via immunohistochemistry (IHC) or immunofluorescence (IF). For immunohistochemistry, slides were deparaffinized by xylenes and rehydrated by ethanol gradient, then heated in a pressure cooker using DAKO retrieval buffer (DAKO, Santa Clara, CA). Endogenous peroxidases were quenched in 3% hydrogen peroxide in methanol for 30 minutes. Tissues were blocked with 0.5% BSA in PBS for 30 minutes and incubated with primary antibodies against: SMAD4, CD3 (Santa Cruz), CD45, or PD-L1 (Cell Signaling) at 1:50–1:200 overnight at 4°C. Slides were developed using HRP-conjugated secondary antibodies followed by DAB substrate/buffer (DAKO).
For immunofluorescence, slides were heated via pressure cooker in DAKO retrieval buffer and tissues blocked with 0.5% BSA in PBS for 1 hour at room temperature. Sections were exposed to primary antibodies against CK19 (University of Iowa Hybridoma Bank), E-Cadherin (Cell Signaling), CD3, (Santa Cruz), or IFNγ (abcam) at 1:50–1:200 overnight at 4°C. Slides were developed using AlexaFluor 488- or 594-conjugated secondary antibodies (1:200–1:1,000, abcam), mounted in DAPI-containing media (Santa Cruz Biotechnology), exposed to DAPI, FITC, and Texas Red filters.
For immunofluorescence, slides were heated via pressure cooker in DAKO retrieval buffer and tissues blocked with 0.5% BSA in PBS for 1 hour at room temperature. Sections were exposed to primary antibodies against CK19 (University of Iowa Hybridoma Bank), E-Cadherin (Cell Signaling), CD3, (Santa Cruz), or IFNγ (abcam) at 1:50–1:200 overnight at 4°C. Slides were developed using AlexaFluor 488- or 594-conjugated secondary antibodies (1:200–1:1,000, abcam), mounted in DAPI-containing media (Santa Cruz Biotechnology), exposed to DAPI, FITC, and Texas Red filters.
10
Histological Analysis of Tissue Sections
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Organs were fixed in 4% formaldehyde (Sigma-Aldrich) for 24 h. The sections were deparaffinized with xylene and rehydrated with decreasing concentrations of ethanol in water. Tissue sections (3-5 µm thick) were stained with hematoxylin and eosin (H&E), periodic acid-schiff (PAS) or subjected to immunohistochemistry (IHC) for cleaved caspase 3 (Asp175; Cell Signaling; 9664; 1:400), SP-C (Santa Cruz Biotechnology, Dallas, TX, USA; sc-13979; 1:50), Ki67 (Ventana Medical Systems, Tucson, AZ, USA; 790-4286; undiluted), or CD31 (Santa Cruz Biotechnology; sc-1506R; 1:1000). Discovery XT IHC research instrument (Ventana Medical Systems) was used for the detection of cleaved caspase 3 and Ki67. An autostainer (Dako, Glostrup, Denmark) was employed for SP-C and CD31. All antibody blocking steps were performed with H 2 O 2 (Dako) and REAL Antibody Diluent (Dako). Conditioning 1 pre-treatment (cell conditioning 1 (CC1), Ventana Medical Systems) was used for the antigen retrieval for subsequent analysis with the Discovery XT systems, and antigen retrieval with EDTA buffer (pH 9) was used for the Dako autostainer. For IHC signal detection, HRP-coupled secondary antibodies were used (Ventana Medical Systems; 760-4311; Jackson ImmunoResearch; 711-065-152) together with DAB substrate buffer (Dako; K3468). Quantification was performed with ImageJ 1.53a (NIH, MD, USA). 36,37
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