MCF10A, HCC1937, HCC1937 BRCA1 and CAL51 cells were cultured alone or transfected with pCDNA3, BRCA1a, BRCA1a Mut#1, BRCA1a Mut#4, BRCA1a Mut#9 or EGFP-Antisense Ubc9 for 24 hours in six-well plates onto glass coverslips overnight. The cells were washed and fixed in icy methanol for 5 minute, and blocked using 10% BSA for 60 min, followed by primary polyclonal Rabbit anti-caveolin-1 antibody 1:150, primary polyclonal anti-SIRT1 antibody (Santa Cruz),l:150 diluted in 1.5% BSA made in PBS at 25°C for 1hr and Alexa 488 goat anti-Rabbit/Alexa 568 goat anti-mouse (Molecular Probes) diluted in 1.5% BSA/PBS for 50 min and stained (Hoechst 33258, Pentahydrate, Life technologies). The cover slips were mounted with Vectashield mounting medium for fluorescence (H-1000 from Vector). The stained cells were examined by LSM 700 Confocal Microscope, equipped with 63× oil Ph immersion objectives. Composite filter cubes were used for the 488–405 as described previously [27 (link)].
Vectashield mounting medium for fluorescence h 1000
Manufactured by Vector Laboratories
Sourced in United States, United Kingdom
Vectashield mounting medium for fluorescence (H-1000) is a glycerol-based medium designed to preserve fluorescence signals in mounted specimens. It is formulated to maintain the integrity and brightness of fluorescent labels.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 488 goat anti rabbit alexa 568 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Rabbit anti caveolin 1 antibody, by Santa Cruz Biotechnology (1 mentions)
Ab139070, by Abcam (1 mentions)
D1306, by Thermo Fisher Scientific (1 mentions)
H 3300, by Vector Laboratories (1 mentions)
Goat anti chicken alexa 488, by Thermo Fisher Scientific (1 mentions)
Polyclonal goat anti mouse immunoglobulins hrp, by Agilent Technologies (1 mentions)
3 protocols using vectashield mounting medium for fluorescence h 1000
1
Immunofluorescence analysis of caveolin-1 and SIRT1
2
Immunofluorescent Labeling of EGFP in Paraffin Sections
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Paraffin samples were cut at 5μm thickness and incubated at 60°C O/N. The next day, they were dehydrated and antigen retrieval was performed with antigen unmasking solution (H-3300, Vector Laboratories, Inc Burlingame, USA) in a pressure cooker for 5 min. Non-specific binding was blocked with PBS/0.1% Tween/1% BSA for 2 h followed by incubation with anti-EGFP (1:200, ab139070, Abcam, Cambridge, UK) at 4°C O/N. The sections were incubated with secondary antibody Alexa Goat anti-chicken 488 (1: 250, Life technologies, Bleiswijk, The Netherlands) and nuclei were stained with Dapi for 5 min (1:3000, D1306, Invitrogen, Bleiswijk, The Netherlands). The sections were mounted with Vectashield mounting medium for fluorescence (H-1000, Vector Laboratories, Inc. Burlingame, USA).
3
Immunofluorescence Staining of α-SMA
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The cells were cultured for 24 h. FGF-1, heparin or SB431542 were then added and the cells were harvested 48 h later. Cells on collagen gel were fixed by paraformaldehyde and permeabilized with 0.1 % Triton Vol. 65 X-100. The unspecific binding was blocked with 1 % FBS for 30 min. The cells were incubated with the antibody to α-SMA (1A4, Sigma) at 4 °C overnight. Polyclonal Goat Anti-Mouse Immunoglobulins/HRP (Dako) as a secondary antibody was applied in dark for 1 h. The nuclei were stained with DAPI. The slides were mounted in Vectashield mounting medium for fluorescence H-1000 (Vector, Peterborough, United Kingdom) and examined by fluorescence microscopy.
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