To further assess the synthesis of the ECM, IHC staining was utilized to detect the aggrecan, collagen type I and II content. After rehydration, tissue sections were blocked by goat serum, treated with hyaluronidase (0.8%) for 20 minutes at 37°C, and then incubated with aggrecan, type I and II collagen antibody (1 : 100, Abcam, UK) for 60 minutes. After washing in PBS, biotinylated secondary antibody (1 : 100, Dako, Denmark) was applied for 30 minutes, washed in PSB, and treated with avidin-biotin complex reagents. Colour was developed using 3,3-diaminobenzidine reagents (Dako, Denmark), and the sections were counterstained with Harris's haematoxylin. The average optical density (AOD) of five randomly selected visual fields (per immunohistochemical slice) under high magnification (400x) was measured using the Image-J analysis system.
3 3 diaminobenzidine reagent
Manufactured by Agilent Technologies
Sourced in Denmark
3,3-diaminobenzidine reagents are a type of laboratory equipment used as a chromogenic substrate in various immunohistochemical and histochemical applications. These reagents produce a brown color reaction when enzymatically catalyzed, allowing for the visualization and detection of target analytes in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Biotinylated secondary antibody, by Agilent Technologies (2 mentions)
Harris hematoxylin, by Thermo Fisher Scientific (1 mentions)
Cytochrome c, by Cell Signaling Technology (1 mentions)
Primary antibodies against cleaved caspase 3, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Merck Group (1 mentions)
4 protocols using 3 3 diaminobenzidine reagent
1
Quantifying ECM Composition via IHC
2
Immunohistochemical Analysis of MIF
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After rehydration, tissue sections were blocked by goat serum, treated with hyaluronidase (0.8%) for 20 minutes at 37°C, and then incubated with MIF antibody (1 : 100, Abcam, UK) for 60 minutes. After washing in PBS, biotinylated secondary antibody (1 : 100, Dako, Denmark) was applied for 30 minutes, washed in PSB, and treated with avidin-biotin complex reagents. Colour was developed using 3,3-diaminobenzidine reagents (Dako, Denmark), and the sections were counterstained with Harris's hematoxylin. The average optical density (AOD) of five randomly selected visual fields (per immunohistochemical slice) under high magnification (400x) was measured using the ImageJ analysis system.
3
Immunohistochemistry Protocol for Tissue Sections
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Formalin-fixed paraffin-embedded tumor or lung tissue were cut to 4 µm sections and IHC was performed as follows: 3% hydrogen peroxide for 10 min, 5% goat serum blocking for 1 h, tissues probed with primary antibody listed in Supplementary Table 1 overnight in 4 °C, secondary streptavidin-conjugated secondary antibody was probed for 1 h, incubation with biotinylated HRP for 30 min, 3,3′-diaminobenzidine reagent (Dako) was used for signal development (~5 min). Slides were counter-stained with Harris Hematoxylin (Thermo Fisher) and further dehydrated and mounted. IHC was performed following Peng et al.7 . Digital images of stained slides were acquired using an Aperio slide scanner AT2 (Leica Biosystems). The Aperio imagescope software version 12.3.3 was used for further analysis.
4
Notch Signaling Pathway Regulation
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AB4 was purchased from Absin Biotechnology Co., Ltd. FBS, DMEM, trypsin and penicillin-streptomycin solution were purchased from HyClone; Cytiva. Primary antibodies against cleaved caspase-3 (#9661), cleaved poly(ADP-ribose) polymerase (PARP) (#5625), cytochrome c (#4280), Notch1 (#4380), Jagged1 (#2620), NICD1 (#4380), hes family bHLH transcription factor 1 (Hes1) (#11988), Ki67 (#9027) and β-actin (#4967), and HRP-labeled secondary anti-rabbit IgG (#7074) were obtained from Cell Signaling Technology, Inc. The hes related family bHLH transcription factor with YRPW motif 1 (Hey1) antibody (#ab11723) was from Abcam. The Annexin V-FITC Apoptosis Detection kit was obtained from EMD Millipore. DAPT, a γ-secretase inhibitor (GSI) that inhibits the Notch pathway, MTT and DMSO were purchased from Sigma-Aldrich; Merck KGaA. The 3,3′-diaminobenzidine reagent was purchased from Dako; Agilent Technologies, Inc.
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