Tissue was sectioned at 5μm or 3μm (for AURKB staining) and allowed to dry. Sections were then deparaffinized and rehydrated prior to heat-induced epitope retrieval in 10mM Tris, 1mM EDTA buffer, pH 9.0, at 100°C in a pressure cooker for 10 minutes. Tissue was allowed to cool for 45 minutes prior to EdU detection or blocking with 10% natural donkey serum in 0.1% Bovine Serum Albumin in PBS (0.1% PBSA) for immunofluorescence staining. Primary antibodies, NKCC1 (1:100 goat SC-21545, 1:250 rabbit CST 85403), Mist1 (1:250 rabbit Abcam ab187978), E-Cadherin (1:250 BD 610181), phospho-histone H3 (1:500 or 1:1000 rabbit Millipore 06–570), aurora-B kinase (1:500 or 1:1000 rabbit Abcam ab2254), and Ki67 (1:500 mouse BD 550609) were applied overnight at 4°C. Secondary antibodies Alexa-fluor 594 α goat (Invitrogen, A11058), Alexa-fluor 594 α rabbit (Invitrogen, A21207), Alexa-fluor 488 α mouse (Invitrogen, A21202), Alexa-fluor 488 α rabbit (Invitrogen, A21207), and Alexa-fluor 488 α goat (Invitrogen, A11055) were applied at a dilution of 1:250 or 1:500 (for Ki67) in 0.1% PBSA and incubated in the dark at room temperature for one hour. DAPI (1:1000, Invitrogen, D1306) was applied for 5 minutes in 0.1% PBSA prior to mounting with Immu-Mount solution (Thermo, 9990402). Slides were allowed to dry overnight at 4°C prior to imaging.
Alexa fluor 594 α rabbit
Manufactured by Thermo Fisher Scientific
Alexa-fluor 594 α rabbit is a fluorescent labeling reagent used for detecting and visualizing rabbit-derived proteins in various biological samples. It is a synthetic dye that emits red fluorescence when excited at the appropriate wavelength, allowing for the specific detection and localization of rabbit target proteins in applications such as immunofluorescence, Western blotting, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 α mouse, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488 α rabbit, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 α goat, by Thermo Fisher Scientific (2 mentions)
E cadherin, by Merck Group (1 mentions)
Immu mount solution, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by AppliChem (1 mentions)
Prolong gold with dapi, by Thermo Fisher Scientific (1 mentions)
Rhmbl, by R&D Systems (1 mentions)
3 protocols using alexa fluor 594 α rabbit
1
Immunofluorescence Staining Protocol for Tissue Sections
2
Immunofluorescence Staining Protocol
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Cells were cultured on normal tissue culture dishes and fixed with 4% paraformaldehyde (Merck) in PBS for 10 min at room temperature, washed with PBS, and permeabilized for 5 min with 0.1% Triton X-100 (AppliChem) in PBS. Blocking was performed for 30 min at room temperature with blocking solution containing PBS-T (0.1% Tween, Sigma-Aldrich) with 3% BSA (Sigma-Aldrich). Primary antibodies (Table S2 ) were diluted into 0.1% BSA in PBS-T and incubated 1 hr at room temperature. After incubation cells were washed three times for 5 min with PBS-T and incubated with the secondary antibodies (1:500, Alexa Fluor-594-α-goat, Alexa Fluor-488-α-mouse, Alexa Fluor-594-α-rabbit, Alexa Fluor-488-α-rabbit, Invitrogen or horse anti-mouse biotin, Vector Laboratories) in PBS-T for 30 min at room temperature. For incubations with secondary biotinylated antibodies, cells were washed three times for 5 min with PBS-T and incubated with Streptavidine 594 (1:500, Invitrogen). The cells were subsequently washed two times for 5 min with PBS and incubated for 15 min with Hoechst (1:15,000, Thermo Scientific). Cells were imaged in PBS.
3
Recombinant Mannose-Binding Lectin Binding Assay
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BHK-21 cells were infected at MOI of 1 with either diluent alone (mock), RRV WT or E2 DM. At 12 hpi, medium was removed and cells were incubated in HBSS with 5 mM CaCl2 with or without 10 µg/ml rhMBL (R&D Systems) for 30 min. Cells were washed with PBS, fixed with PFA, and stained using standard techniques using the following antibodies: α-MBL-C (SCBT 1:50); α-RRV (ATCC 1:1000); Alexa Fluor 488-α mouse (Invitrogen 1:1000); and Alexa Fluor 594 α-rabbit (Invitrogen 1:1000). Slides were mounted with ProLong Gold with DAPI, (Invitrogen) and imaged by fluorescence microscopy. Images were processed using ImageJ (NIH).
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