UM004 xenografts were excised from mice following seven days of treatment with vehicle, abemaciclib, merestinib, or in combination, fixed in formalin overnight and then embedded in paraffin. After sectioning, paraffin-embedded tissue sections were deparaffinized and antigen retrieval was accomplished using high pH conditional buffer. Sections were incubated with anti-phospho-cMET, phospho-RB, FOXM1, and Ki67 antibody overnight. On the following day, sections were incubated for 30 min in ImmPRESS AP Reagent (Vector Laboratories, Burlingame, CA, USA), followed by incubating for 5 to 10 min in ImmPact Vector Red (Vector Laboratories). Sections were counterstained with hematoxylin. Immunohistochemistry analysis was performed in three tumors from each group.
Immpress ap reagent
Manufactured by Vector Laboratories
Sourced in United States
The ImmPRESS AP Reagent is a horseradish peroxidase (HRP) conjugated polymer detection system designed for immunohistochemical staining. It provides a sensitive and reliable method for localizing antigens in tissue sections.
Automatically generated - may contain errors
4 protocols using immpress ap reagent
1
Immunohistochemical Profiling of Xenograft Tumors
2
Histopathological Analysis of Tumor Tissues
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For histopathological evaluation, hematoxylin and eosin (H&E) staining was performed on paraffin-embedded tumor tissue sections. For immunohistochemistry, sections were stained with primary antibodies overnight at 4°C. On the next day; sections were incubated for 30 minutes in ImmPRESS AP Reagent (Vector Laboratories, Burlingame, CA), followed by incubating for 2–15 minutes in ImmPACT NOVA-RED (Vector Laboratories). The following primary antibodies were purchased from Agilent (Santa Clara, CA): cancer antigen 19-9 (CA19-9), carcinoembryonic antigen (CEA), cytokeratin 20 (CK20), and Ki-67 [9 (link)]. For terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, In Situ Cell Death Detection Kit (Roche, Penzberg, Germany) was used, followed by the manufacturer’s instruction. Ki-67 positive area or TUNEL positivity in immunohistochemistry (IHC) image was measured by ImageJ software (National Institutes of Health, Bethesda, MD).
3
Immunohistochemistry for Melanoma Markers
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For immunohistochemistry, 5 μm tissue sections were steamed for 20 min with antigen retrieval solution and stained with primary antibodies, SOX10 (A-2, sc-365692; Santa Cruz Biotechnology, Santa Cruz, CA), HMB45 (M0634; Dako, Carpentaria, CA) and S100 (Z0311; Dako, Carpentaria, CA) overnight at 4 °C. On the next day, sections were incubated for 30 min in Imm-PRESS AP Reagent (Vector Laboratories, Burlingame, CA, USA), followed by 5 min incubation with ImmPACT NOVA-RED (Vector Laboratories).
4
Histopathological Evaluation of Pigmented Tumor Tissues
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For histopathological evaluation, H&E staining was performed on paraffin-embedded tumor tissue sections. The degree of pigmentation of tissue samples was assessed based on the Collaborative Ocular Melanoma Study (COMS) classification system [49 (link)]. For immunohistochemistry, sections were stained with primary antibodies overnight at 4 °C. On the next day; sections were incubated for 30 min in ImmPRESS AP Reagent (Vector Laboratories, Burlingame, CA, USA), followed by incubating for 2–15 min in ImmPACT NOVA-RED (Vector Laboratories). The following primary antibodies were purchased from Agilent (Santa Clara, CA, USA): Melanosome (Clone HMB45), Melan-A, S100, and Ki67. All sources of specific antibodies for these markers and their dilution rates have been previously described [42 (link)] except for Ki67 (dilution rate; 1:50). Staining data for Ki67 were quantitated by counting all positive nuclei per field of vision at ×400 magnification using ImageJ software (available at http://rsb.info.nih.gov/ij ) [40 (link)].
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