Full staining and antibody detail provided as Supplemental Material . Briefly, 4% paraformaldehyde-fixed paraffin sections were rehydrated, unmasked and blocked in TBS/0.1% Triton X-100 containing 1% BSA. Primary antibodies were incubated at 4C overnight in blocking buffer. For immunohistochemistry, sections were incubated with anti-rabbit ImmPRESS HRP-conjugated secondary antibodies (Vector Laboratories, #MP7401) and chromagen development performed using ImmPact DAB (Vector Laboratories, #SK4105). Stained slides were counterstained with Harris’ hematoxylin. For immunofluorescent stains, secondary antibodies were applied in TBS for 1 hour at room temp and sections counterstained with DAPI. In situ hybridization for Olfm4 and Lgr5 were performed essentially as previously described (Gregorieff et al., 2005 (link)) (van der Flier et al., 2009 (link)).
Immpress hrp conjugated secondary antibody
Manufactured by Vector Laboratories
ImmPRESS HRP-conjugated secondary antibodies are horseradish peroxidase (HRP)-labeled antibodies used for immunohistochemistry and immunocytochemistry applications. They are designed to bind and detect primary antibodies in these assays.
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4 protocols using immpress hrp conjugated secondary antibody
1
Multimodal Profiling of Intestinal Stem Cells
2
Immunohistochemistry and Immunofluorescence Protocols
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Individual antibody details are listed in suppl. Table 2. Human paraffin-embedded sections were deparaffinised, rehydrated and subjected to epitope retrieval. Sections were blocked with 10% normal horse serum (NHS) followed by overnight incubation with primary antibody and then incubated with ImmPRESS HRP-conjugated secondary antibodies (Vector Laboratories). Slides were visualized with ImmPACT-DAB (Vector Laboratories) as the chromogen. Sections were counterstained with haematoxylin and DePex mounted. Dual colour IHC was performed sequentially. After detection of the primary antibody with ImmPACT-DAB, incubation with the second primary antibody was conducted and detected using the ABC-alkaline phosphatase detection system, using Vector Blue as substrate. When using snap-frozen tissue for IHC, slides were pre-treated with cold methanol and then followed the same steps as for paraffin tissue.
For immunofluorescence, sections were fixed with 100% methanol at − 20 °C, blocked and incubated overnight with primary antibodies. Sections were incubated with the appropriate secondary antibody conjugated to a fluorochrome, as described previously [35 (link)] and nuclei counterstained with DAPI (Sigma-Aldrich) and mounted with Vectashield Antifade Mounting Media (Vector Laboratories).
For immunofluorescence, sections were fixed with 100% methanol at − 20 °C, blocked and incubated overnight with primary antibodies. Sections were incubated with the appropriate secondary antibody conjugated to a fluorochrome, as described previously [35 (link)] and nuclei counterstained with DAPI (Sigma-Aldrich) and mounted with Vectashield Antifade Mounting Media (Vector Laboratories).
3
Immunohistochemical Analysis of Lymphoma Tissue
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We performed immunohistochemistry (IHC) staining of primary lymphoma tissues on a tissue microarray (TMA) slide (US Biomax, LM801) for p-eIF4E1 staining at Mass Histology Services (Boston, MA). In brief, TMA slide was de-waxed and hydrated. Slide was then pre-treated with citrate buffer and rinsed in water. Slide was treated with 3% H2O2 followed by another rinse in water and two washes in PBS. Sample was then blocked in 2% horse serum and incubated in a primary antibody at 1:250 dilution for 1 h at room temperature. After three washes, the slide was incubated with an ImmPRESS HRP conjugated secondary antibody (Vector Labs) for 45 min at room tenperature. Following two more washes in PBS, slide was treated with DAB substrate and rinsed in water. Slide was then counterstained with haematoxylin and differentiated in acid alcohol (2 quick dips). Finally, the slide was placed under running water for 10 mins, dehydrated and mounted with coverslip.
4
Immunohistochemistry Staining of Lymphoma Tissues
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