Alexa fluor 633 conjugated goat anti rabbit igg

Oocytes were fixed in 4% PFA (30 min, RT), permeabilized with 0.5% Triton-X100 (30 min, RT) and blocked with 3% BSA. Calnexin, an ER marker, was labelled with a rabbit polyclonal antibody (1:200, Abcam) followed by an Alexa Fluor 633-conjugated goat anti-rabbit IgG (1:200; Invitrogen, Thermo Fisher Scientific) and β-tubulin – with a mouse monoclonal antibody conjugated with FITC (1:50). Embryos were incubated in the primary antibodies overnight at 4 °C, washed in PBS and 3% BSA and then, if required, incubated with the secondary antibody for 2 hrs in RT. DNA was stained with propidium iodide (0.01 mg/ml in PBS; 30 min, RT or overnight, 4 °C). Oocytes were analysed on an inverted confocal microscope (Zeiss and Olympus).

After fixation and permeabilization, we blocked the FLSs with 5% bovine serum albumin (BSA), then incubated the cells in phosphate-buffered saline (PBS) containing rabbit anti-human polyclonal antibody to CYLD (Abcam, USA) or normal rabbit IgG (control) overnight at 4 °C. The secondary antibody (red) was Alexa Fluor 633 conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA), used at a 1:1000 dilution for 1 h at 37 °C. We used 4,′6-diamindino-2-phenylindole (DAPI; Sigma-Aldrich) to stain the cell nuclei (blue) at a concentration of 1.43 μM for 3 min and mounted ProLong Gold Antifade Reagent (P36934; Invitrogen) to the coverslips. Images were examined and analyzed with a 160 Zeiss LSM 510 confocal microscope (Carl Zeiss AG, Jena, Germany).

To detect GH5 and GH6 catalytic modules in tissues, slide-mounted sections were deparaffinized as described in [28 ] then incubated in 10 mM sodium citrate buffer, pH 6.0 with 0.05% Tween 20 for 4 h at 82°C and then 2 h at room temperature to promote protein refolding. Slides were rinsed briefly with TBS-T buffer and blocked with 5% skimmed milk in TBS-T buffer overnight at 4°C then incubated with 4 µg ml⁻¹ primary antibody or 1 : 200 pre-immune serum in 1% BSA in TBS-T buffer for 2 h at room temperature. To assess non-specific binding of the secondary antibody, control slides were prepared without primary antibody and with pre-immune serum respectively. Slides were then washed 3× with TBS-T for 3 min each, followed by incubation with 5 µg ml⁻¹ Alexa Fluor 633-conjugated goat anti-rabbit IgG (ThermoFisher Scientific A-21071) for 1 h in the dark at room temperature, then washed 3× with TBS-T buffer for 3 min each and air-dried. Slides were mounted using ProLong Diamond Antifade Mountant with DAPI (ThermoFisher P36966) and allowed to cure for 48 h. Sections were imaged on Zeiss LSM 880 confocal microscope (Beth Israel Deaconess Medical Center or Institute for Chemical Imaging of Living Systems, Northeastern University).

Sections were incubated successively with 10% NGS in PBS containing 0.3% Triton X-100 (PBS-X) for 30 min, 1:2,000 diluted anti-fluorogold polyclonal antibody (AB153-I; EMD Millipore, Billerica MA) in PBS-XG for 48 h, and 4 μg/ml Alexa Fluor 633-conjugated goat anti-rabbit IgG (A-21071; Thermo Fisher Scientific) in PBS-XG for 2 h. Sections were counterstained with a fluoro-Nissl solution for 60 min, washed with PBS, mounted, and coverslipped.

MCL cells were cytospun, fixed in 4% paraformaldehyde and permeabilized in 0.5% Triton-X100. The slides were then stained with an anti-cyclin D1 Ab (sc-718, Santa Cruz Biotechnology), and then with Alexa Fluor 633-conjugated goat anti-rabbit IgG (in green) and counterstained with DAPI (4′,6-diamidino-2-phenylindole dihydrochloride, Molecular Probes, in blue). Slides were observed with a confocal fluorescence microscope (Fluoview FV 100, Olympus). For XPO1 inhibition, cells were treated with KPT-330 for 4 h before IF analysis.
Cultured cells were either left untreated or treated with 200 ng/ml SDF1 to stimulate chemotaxis. Fixed and permeabilized cells were incubated with rhodamine-phalloidin, as recommended by the supplier (Molecular Probes) and analysed by fluorescence microscopy. In some experiments, SDF1-treated cells were also treated with 300 nM KPT-330 (or vehicle) for 4 h before F-actin staining.

To immunostain for MLC2 phosphorylated at Ser19, the cells were fixed with 10% formaldehyde in phosphate-buffered saline (PBS) supplemented with 0.1 M HEPES pH 7.4 and then permeabilized with 0.2% Triton X-100. After blocking with 5% goat serum in PBS, the cells were incubated with the anti-pMLC2 (Ser19) antibody. Alexa Fluor 633-conjugated goat anti-rabbit IgG (Molecular Probes) was used as a secondary antibody. Alexa Fluor 488 phalloidin (Molecular Probes) and DAPI (Vector Laboratories) were used to stain F-actin and nuclei, respectively. To visualize p53 and F-actin simultaneously in single cells within spheroids, cells cultured overnight on the 2 kPa substrate were transfected with the Lifeact-GFP expression vector (a gift from Roland Wedlich-Söldner, University of Munster, Munster, Germany [30 (link)]) together with the control or HA-tagged p53 expression vector. After 24 h, the cells were treated with doxorubicin for 16 h. The cells were fixed with 10% formaldehyde in PBS containing 0.1 M HEPES pH 7.4. Doxorubicin incorporated into the cells was visualized using its autofluorescence (e.g., ex: 488 nm/em: >580 nm [31 (link)]). Images were acquired using a confocal microscope (LSM700; Zeiss) and then analyzed with ImageJ software (NIH).