Three-micrometres sections made from formalin-fixed paraffin embedded tissues were immunostained using Dako Envision™ FLEX+ system (K8012; Dako, Glostrup, Denmark) and the Dako Autostainer. Paraffin sections were deparaffinized and epitopes unmasked in PT-link with low pH target retrieval solution (Dako), and then blocked with peroxidase blocking (Dako) for 5 min. The slides were incubated at 4 °C overnight with mouse anti-human ALDH1 antibody (1: 3000, 83 ng IgG1/ml, Clone 44, Lot. No. 03817, BD Transduction Laboratories™), followed up by incubation with mouse linker for 15 min and HRP for 30 min at room temperature. Slides were then stained with 3, 3′-diaminobenzidine tetrahydrochloride (DAB) for 10 min and counter-stained with hematoxylin, dehydrated, and mounted in Richard-Allan Scientific Cyto seal XYL (Thermo Scientific, Waltham, MA, USA). Known ALDH1-positive human vulvar squamous cell carcinoma slide [28 ] was used as positive control. Mouse myeloma protein of the same subclass and concentration as the primary mouse anti-ALDH1 antibody was used for negative control.
Peroxidase blocking
Manufactured by Agilent Technologies
Sourced in Denmark, United States
Peroxidase blocking is a laboratory technique used to inhibit endogenous peroxidase activity in tissue samples. This method helps prevent non-specific staining during immunohistochemical or in situ hybridization analyses.
Automatically generated - may contain errors
Lab products found in correlation
Envision flex system, by Agilent Technologies (4 mentions)
Aec 3 amino 9 ethyl carbazole chromogen, by Thermo Fisher Scientific (4 mentions)
Ultravision lp detection system, by Thermo Fisher Scientific (4 mentions)
Cytoseal xyl, by Thermo Fisher Scientific (3 mentions)
Autostainer, by Agilent Technologies (2 mentions)
Protein block, by Agilent Technologies (2 mentions)
Ki 67 antibody, by Agilent Technologies (2 mentions)
Pt link pre treatment module, by Agilent Technologies (2 mentions)
Autostainer link 48 device, by Agilent Technologies (2 mentions)
Envision flex kit, by Agilent Technologies (2 mentions)
Low ph target retrieval solution, by Agilent Technologies (1 mentions)
Zenon alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Anti runx2, by Abcam (1 mentions)
Anti osx, by Abcam (1 mentions)
Anti sost, by Abcam (1 mentions)
Anti mef2c, by Abcam (1 mentions)
Anti il 1β, by Abcam (1 mentions)
Anti alp, by Abcam (1 mentions)
Anti hif 1α, by Abcam (1 mentions)
Dm4000b microscope, by Leica (1 mentions)
19 protocols using peroxidase blocking
1
Immunohistochemical Detection of ALDH1 in FFPE Tissues
2
Standardized Immunohistochemistry Protocol
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The Dako Envision FLEX+ system (K8012; Dako, Glostrup, Denmark) and the Dako Autostainer were used for IHC. Paraffin sections were deparaffinized and epitopes unmasked in PT link with high pH/low pH target retrieval solution (Dako), and then blocked with peroxidase blocking (Dako) for 5 minutes. The slides were incubated with primary antibody 30 minutes at room temperature, following up with rabbit/mouse linker (Dako) according to the resource of primary antibody for 15 minutes and HRP for 30 minutes at room temperature. Slides were then stained with 3, 3′-diaminobenzidine tetrahydrochloride (DAB) for 10 minutes and counter-stained with hematoxylin, dehydrated, and mounted in Richard-Allan Scientific Cyto seal XYL (Thermo Scientific, Waltham, MA, USA). Already known each antibody positive tissue was used as positive control in the same procedure. The same positive control slide was used as a negative control incubated with the same concentration of non-immune rabbit/mouse IgG replacing the primary antibody. The information for each primary antibody is shown in Table 1 .
3
Phosphorylated ERK and CREB Immunohistochemistry
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Paraffin sections of the SMG (4-μm thick) were deparaffinized in xylene and rehydrated in graded alcohol series. Sections were immersed in citrate buffer pH 6.0 (Epitomics) at 97°C for 20 min, washed in PBS and immersed in peroxidase blocking (DAKO) for 10 min. The samples were then washed in PBS and incubated in protein block (DAKO) for 1 h. The first antibody phosphorylated ERK (1:150, Cell Signaling Technology, Cat# 4370, RRID:AB_2315112) was prepared with Zenon Alexa Fluor 594 (Molecular Probes, Inc., Eugene, OR) according to manufacturer’s instructions, and applied to the samples and incubated in a humidified dark chamber for 2 h. Then the sections were washed in PBS for 25 min and subsequently incubated with the second antibody phosphorylated CREB (1:150, Abcam Cat# ab32096, RRID:AB_731734) prepared with Zenon Alexa Fluor 488 for 2 h. DAPI was used for nuclear counterstaining.
4
Immunohistochemical Analysis of Bone Markers
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Sections of jaw and femur tissues were subjected to antigen retrieval (pH 6.0, Abcam, Tokyo, Japan), followed by peroxidase blocking (DAKO, Santa Clara, CA, USA). Anti-ALP (1:100, a gift from Hokkaido University, Hokkaido, Japan), anti-Runx2 (1:125, Abcam, Tokyo, Japan), anti-OSX (1:75, Abcam, Tokyo, Japan), anti-Mef2c (1:500, Abcam, Tokyo, Japan), anti-SOST (1:50, Abcam, Tokyo, Japan), anti-IL-1β (1:100, Abcam, Tokyo, Japan) and anti–HIF–1α (1:100, Abcam, Tokyo, Japan) were used as primary antibodies. The specimens were exposed to the same DAB reaction conditions, and all images were captured using a microscope (OLYMPUS, Tokyo, Japan).
5
Immunohistochemical Analysis of Piwi-like 1 Protein
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For the Piwi-like 1 protein expression study, a manual immunohistochemistry (IHC) protocol was applied as previously described26 (link). Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), a primary antibody against Piwi-like 1 (polyclonal goat IgG, N-17; Cat.-No. 22685; Santa Cruz, Heidelberg, Germany) was applied for 30 min. The stained specimens were viewed at an objective magnification of x100 and x200 by an experienced uropathologist (AH). The expression of Piwi-like 1 was detected in the cytoplasm by assessing the percentage of stained tumor cells and the staining intensity semi-quantitatively but it was not detected in the normal tissue adjacent to the tumor (Suppl. Fig. 1 ). Their product resulted in an immunoreactive score (IRS) from 0 to 1227 (link). Negative control slides without the addition of primary antibody were included for each staining experiment. For the survival analysis, the patients were grouped by IRS = 0 and IRS > 0. Photos were taken with a Leica DM 4000B microscope with 20x HC PL Fluotar objective (Leica, Wetzlar, Germany) and with a Jenoptik Gryphax Arktur camera (Jenoptik AG, Jena, Germany). A white balance was performed.
6
Immunohistological Analysis of Fabricated Cell Sheets
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Immunohistology was performed according to methods described in our previous article30 (link). Fabricated cell sheets were fixed in 4% paraformaldehyde (Wako Pure Chemical Industries), dehydrated, embedded in paraffin, sectioned (4 μm) and mounted on glass slides. The slides were de-paraffinized, and antigen retrieval was performed by autoclaving with citrate buffer (Histo-VT One, Nacalai Tesque). Peroxidase blocking (Dako) and protein blocking (Nacalai Tesque) were carried out to prevent non-specific reactions. The slides were then incubated overnight at 4 °C in PBS containing primary antibody (Table S2 ). After washing with PBS, the slides were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (EnVision Detection System, Peroxidase/DAB, Rabbit/Mouse, HRP; K5007; Dako) at room temperature for 1 h. The sections were washed again and treated with 3,3′-diaminobenzidine (K5007; Dako) to visualize the HRP. Nuclei were stained by hematoxylin. De-paraffinized sections were also stained with hematoxylin and eosin (HE) for histological evaluation.
7
Multimodal Liver Tissue Analysis
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For Oil Red O staining, 6-µm thin cryosections were mounted on poly-l -lysine-coated slides and dried overnight. Working solution of Oil Red O was prepared by mixing stock solution [0.5% (w/v) in 2-propanol] 3:2 with water followed by filtration. Working solution was incubated for 10 min. To stain liver glycogen, formalin-fixed, paraffin-embedded livers were cleared and incubated in 1% periodic acid (25 min) followed by Schiff reagent (Sigma-Aldrich, St. Louis, MO, USA; 25 min) and rinsing in SO2 water. Immunohistochemical staining of formalin-fixed, paraffin-embedded livers was performed after antigen retrieval (120°C, 7 min at pH 9) and peroxidase blocking (Dako, Glostrup, Denmark) using the UltraVision LP detection system (Thermo Fisher Scientific) according to the manual with 1 ng/µl Ki-67 antibody (M728; Dako). For color reaction, AEC (3-amino-9-ethylcarbazole) chromogen (Thermo Fisher Scientific) was used. Stainings were performed in a LabVision 2D autostainer (Thermo Fisher Scientific). Mouse IgG1 was used as negative control. Counterstaining with hematoxylin was done on all slides. All quantifications were performed by Ilastik Interactive Learning and Segmentation software (http://ilastik.org/ ). Automatic counting of segments was done by a customized R script (R Foundation for Statistical Computing, Vienna, Austria; http://www.r-project.org/ ).
8
Immunohistochemical Detection of Liver MPC1 and MPC2
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IHC detection of mouse liver MPC1 and MPC2 was performed with the Dako Envision FLEX+ system (K8012; Dako, Glostrup, Denmark). Paraffin slices were de-paraffinized and dehydrated in xylene and ethanol series, respectively. Epitope unmasking was performed with microwaving antigen retrieval in citrate buffer (pH 6.0) for 15 minutes before being returned to room temperature and washed in PBS. Unspecific blocking was operated by peroxidase blocking (Dako) for five minutes. The sections with primary antibody (BRP44L, 1:700; BRP44, 1:500) were incubated at 4°C for 14h. The second antibody and HRP were used for 15 minutes and 30 minutes in sequence. Diaminobenzidine tetrahydrochloride (DAB) was used to stain the slides for 10 minutes and counterstained with hematoxylin, dehydrated and mounted.
9
Immunohistochemical Analysis of COX-2 Expression
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Cells were seeded in 8-wells chambers (50,000 cells/well). Next day, medium was replaced and cells were stimulated during 24 h as above. After 15 min fixation with paraformaldehyde 4% and peroxidase blocking (Dako, Copenhagen, Denmark), cells were incubated 2 h with COX-2 antibody and 1 h with secondary HRP anti-rabbit antibody. Development of the peroxidase staining was performed with DAB and visualized in Leica DM IL LED microscope, using Leica Application Suite (Solms, Germany).
10
Immunohistochemistry Protocol for GLUD1 and GLS1
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The IHC was performed using the EnvisionTM+System. Shortly, sections were deparaffinized by PT-Link machine for 1 hour and 20 minutes and blocked with peroxidase blocking (Dako) for 5 minutes after rinsing by DAKO wash buffer. Then the slides were incubated with the primary antibody at 4°C overnight followed with mouse or rabbit linker for 15 minutes and HRP for 30 minutes at room temperature. The slides were then stained with 3,3′-diaminobenzidine tetrahydrochloride (DAB, Dako) for 5 minutes, counter-stained with hematoxylin, dehydrated and mounted in Richard-Allan Scientific Cytoseal XYL (Thermo Scientific, Waltham, MA, USA). The corresponding non-immune rabbit IgG and non-immune mouse IgG were used as negative controls for GLUD1 and GLS1, respectively.
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