The expression of HDAC1 protein within tissues were verified by IHC as previously described (6 (link)). A total of 8 cases of tissue (4 cases in medulloblastoma and 4 cases in AT/RT) used to verify HDAC1 protein expression. Of these, 3 cases of each group were newly obtained, and 1 case of each group was included in the previous RT-qPCR analysis. Briefly, the sections were incubated with primary antibodies, HDAC1 (1:1000, Abcam, Cambridge, MA), for 32 min at 37°C, and a secondary antibody for 20 min at 37°C. The stained sections were detected using the Ventana ChromoMap Kit (Ventana Medical Systems) and discovered using XT automated IHC strainer (Ventana Medical Systems, Oro Valley, AZ).
Ventana chromomap kit
Manufactured by Roche
Sourced in United States
The Ventana ChromoMap Kit is a laboratory product designed for chromogenic in situ hybridization (CISH) analysis. It provides reagents and materials required to detect and visualize specific DNA or RNA sequences within tissue samples. The kit enables researchers and clinicians to analyze the presence, distribution, and characteristics of targeted genetic markers in a variety of specimen types.
Automatically generated - may contain errors
Lab products found in correlation
Discovery xt, by Roche (5 mentions)
Hdac1, by Abcam (1 mentions)
Axioskop light microscope, by Zeiss (1 mentions)
Mab4399, by Merck Group (1 mentions)
Ventana omnimap anti mouse secondary antibody, by Roche (1 mentions)
Ultramap anti rb hrp, by Roche (1 mentions)
Imagescope, by Leica (1 mentions)
Aperio digital pathology, by Leica (1 mentions)
6 protocols using ventana chromomap kit
1
Immunohistochemical Analysis of HDAC1 Expression
2
Immunohistochemical Analysis of CD44 and CD24 Expression
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This study evaluated the expression of two proteins in tumor tissues: CD44 and CD24. The following antibodies were used: Rabbit monoclonal anti-CD44 (1:600; Boster Biological Technology Co., Ltd.; cat. no. PA1021-2) and mouse monoclonal anti-CD24 (1:50; Abcam; cat. no. MA5-11833). Using the Discovery XT automated IHC instrument (Ventana Medical Systems, Inc.), the sections were stained using the following procedures. Firstly, detection was performed using a Ventana Chromo Map kit (Ventana Medical Systems, Inc.). Sections were deparaffinized using an EZ Prep solution included in the Chromo Map kit. CC1 standard (Tris, borate and EDTA buffer; pH 8.4) was used for antigen retrieval (at 95°C for 44 min). Treatment with Inhibitor D (3% H2O2) for 4 min at 37°C was used to block endogenous peroxidase. Sections were then incubated with primary antibodies for 32 min at 37°C and with an OmniMap anti-mouse secondary antibody (Ventana Medical Systems, Inc.; cat. no. 760-4310) for 20 min at 37°C. Sections were incubated in 3,3′-diaminobenzidine + H2O2 substrate for 8 min at 37°C, followed by hematoxylin and eosin reagent counterstain for 2 min at 37°C. Reaction buffer (Tris buffer; pH 7.6) was used as a washing solution. Slides were evaluated on a Zeiss Axioskop light microscope (Carl Zeiss) equipped with Zeiss Plan-Neofluar objective lenses (×40, ×200).
3
Immunohistochemical Analysis of CD133 in FFPE Tissues
4
Automated IHC Staining for Tissue Sections
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Formalin-fixed, paraffin-embedded tissue was sectioned and placed on slides. Sections were stained using the Discovery XT automated immunohistochemistry (IHC) stainer (Ventana Medical Systems; Tucson, AZ, USA) and Ventana Chromo Map Kit (Ventana Medical Systems) according to the manufacturer’s instructions. The sections were deparaffinized using EZ prep solution; the antigen was retrieved in cell a conditioning solution (CC1; Ventana Medical Systems) under standard conditions (100 °C, 60 min), and endogenous peroxidase was inhibited by treatment with 3 % H2O2 for 4 min. Then, the sections on slides were incubated with the primary antibody that had been used in the immunoblot analysis (dilution 1:300 for anti-HNRNPA1 and anti-CEA and dilution 1:100 for other antibodies) for 60 min at 37 °C, and then with a secondary antibody (UltraMap anti-RB HRP or UltraMap anti-MS HRP; Ventana Medical Systems) for 28 min at 37 °C. The sections were incubated in diamidobenzidine and H2O2 for 8 min at 37 °C followed by counterstaining with hematoxylin and treatment with bluing solution. On completion of staining, sections were dehydrated in alcohol, cleared in xylene, and mounted in synthetic resin.
5
Immunohistochemical Analysis of cIAP2 in Gastric Tissues
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Tissues from the antrum were fixed in 10% neutral buffered formalin, paraffin-embedded, and then cut into 4-μm sections. Slides were stained using the Discovery XT automated immunohistochemistry stainer (Ventana Medical Systems Inc., Tucson, AZ, USA) and detection was performed using the Ventana Chromo Map Kit (Ventana Medical Systems). Sections were deparaffinized using the EZ Prep solution. CC1 (pH 8.4 buffer containing Tris/Borate/EDTA) was used for antigen retrieval. Endogenous peroxidases were blocked with Inhibitor D (3% H2O2) for 4 minutes at 37°C temperature. Slides were incubated with primary antibodies for 32 minutes at 37°C and a secondary antibody for 20 minutes at 37°C. Slides were incubated in diaminobenzidine (DAB) plus H2O2 substrate for 8 minutes at 37°C followed by hematoxylin and bluing reagent counter-stain at 37°C. Reaction buffer (pH 7.6 Tris buffer) was used as a washing solution. A monoclonal antibody for cIAP2 (Santa Cruz Biotechnology; Santa Cruz, CA, USA; dilution 1:30) was used as a primary antibody, and biotin-labeled anti-mouse immunoglobulin G (UltraMap anti-Ms HRP Roche; Ventana Medical Systems, Inc.) was used as a secondary antibody. Negative controls were treated similarly while excluding the primary antibodies. The whole slide area was assessed and positive staining was qualitatively evaluated by an experienced pathologist (E.S.).
6
Quantifying Pancreatic Islet P62 Staining
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Paraffin blocks embedding pancreas from the previous study [10 (link)] were cut and placed on slides. The slides were stained using the Discovery XT automated immunohistochemistry stainer (Ventana Medical Systems Inc., Tucson, AZ, USA). Detection was done using the Ventana ChromoMap Kit (Ventana Medical Systems). The stained sections were analyzed using Aperio Digital Pathology and ImageScope (Leica Biosystems, Wetzlar, Germany). About 10.1±1.8 islets were counted in every mouse for the measurement of P62 statin intensity, and quantification of the intensity per islet area was calculated using ImageJ (National Institutes of Health, Bethesda, MD, USA).
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