Tumor tissues were fixed with 4% paraformaldehyde, and then dehydrated and embedded in paraffin. From the rehydrated tissue slices, antigen retrieval was performed using the heat-induced antigen retrieval in citrate buffer (Vector Laboratories, CA). The tissues were made permeable with PBS, containing 0.2% TritonX-100, and then treated with 3% hydrogen peroxide to inactivate the endogenous peroxidase. The tissues were washed with PBS, containing 0.05% Tween-20, and then incubated for 2 h with a blocking solution (MOM blocking buffer, Vector Laboratories, CA). The samples were then incubated with CD44 antibody overnight at 4 °C, followed by incubation with secondary antibodies for 1 h at room temperature. Signals were amplified using an ABC kit (Vector Laboratories) and visualized using a 3,3′-diaminobenzidine substrate kit (SK-4105, Vector Laboratories). The tissues were further stained with H&E, dehydrated, and mounted (H5000, Vector Laboratories).
Mom blocking buffer
Manufactured by Vector Laboratories
MOM blocking buffer is a component used in various laboratory techniques. It serves to block non-specific binding sites, preventing undesired interactions during the analysis or detection process. The specific formulation and function of this buffer are detailed in the product documentation provided by the manufacturer.
Automatically generated - may contain errors
Lab products found in correlation
Abc kit, by Vector Laboratories (2 mentions)
H 5000, by Vector Laboratories (2 mentions)
Citrate buffer, by Vector Laboratories (2 mentions)
3 3 diaminobenzidine substrate kit, by Vector Laboratories (1 mentions)
Alexa fluor 488 anti mouse f4 80, by BioLegend (1 mentions)
Ki 67 primary antibody, by BD (1 mentions)
3 protocols using mom blocking buffer
1
Immunohistochemical Analysis of CD44 Expression
2
Comprehensive Inflammatory Cell Profiling
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All stains were performed on formalin-fixed paraffin-embedded skin sections. Inflammatory infiltrates were initially assessed by H&E staining. For cell-specific stains, sections were deparaffinized and slides boiled in sodium citrate buffer as described previously. For CD3 staining, rabbit anti-CD3 antibody (Abcam, Cambridge, United Kingdom, ab5690, 1:150) and an EnVisionþ System-HRP (DAB) kit (Dako, Carpinteria, CA, K4011) were used according to the manufacturer's instructions. For staining of other inflammatory cell markers, sections were treated with MOM blocking buffer (Vector Laboratories, Burlingame, CA, PK2200) at room temperature for 1 hour before incubation separately with primary antibodies diluted in blocking buffer (Alexa Fluor 488 anti-mouse Ly-6G, 1:400, Biolegend, San Diego, CA, 108419; Alexa Fluor 488 anti-mouse F4/80, 1:600, Biolegend 123119; Alexa Fluor 594 anti-mouse CD272 (BTLA), 1:200, Biolegend 139111) overnight at 4 C. After washing with PBS containing 0.05% Tween-20, ProLong Gold Antifade Mountant with DAPI was applied and slides were stored in the dark at 4 C.
Methods for staining of mast cells and Trichrome staining are provided in Supplementary Materials and Methods.
Methods for staining of mast cells and Trichrome staining are provided in Supplementary Materials and Methods.
3
Ki67 Expression Quantification in Xenograft Tumors
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Immunohistochemistry was done as described previously [19 (link)]. Xenograft tumor tissues were fixed with 4% PFA, dehydrated, and embedded in paraffin. The rehydrated tissue slices were treated by heat-mediated antigen retrieval in citrate buffer (Vector Laboratories, Burlingame, CA). The tissues were made permeable with PBS containing 0.2% TritonX-100, then treated with 3% hydrogen peroxide to inactivate endogenous peroxidase. The tissues were washed with PBS containing 0.05% Tween-20, and incubated for 2 h with blocking solution (MOM blocking buffer, Vector Laboratories, CA). Primary Ki67 antibody (550609, BD Biosciences, San Diego, CA, dilution, 1:2000) was incubated overnight at 4°C, and secondary antibodies were incubated for 1h at room temperature. Signals were amplified with ABC kit (Vector Laboratories) and visualized with 3,3′-diaminobenzidine substrate kit (SK-4105, Vector Laboratories). The tissues was further stained with hematoxylin, then dehydrated, and mounted (H5000, Vector Laboratories). Photograph was taken and Ki67 positive cell percentage was calculated.
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