Prolong gold antifade solution

Only blastocyst stage embryos at time of collection were processed for immunostaining as previously described (Ross et al., 2008 (link)). Embryos were washed with phosphate buffered saline (PBS; GIBCO) containing 1 mg/mL PVA (PBS-PVA) three times and then fixed in 4% paraformaldehyde containing 1 ml/mL PVA for 15 min at room temperature. After washing three times with PBS-PBA, blastocysts were permeabilized with PBS-PVA containing 1% Triton X-100 for 30 min, washed in PBS-PVA containing 0.1% Triton X-100 (washing buffer; WB), and blocked in PBS-PVA supplemented with 10% normal donkey serum. Embryos were incubated with primary antibodies (rabbit anti-SOX2 antibody, BioGenex Cat# NU579-UC and mouse anti-human nuclei antibody, Millipore Cat# MAB1281) overnight at 4°C. After repeated washes in WB, the embryos were incubated with secondary antibodies (Alexa Fluor 568 anti-rabbit IgG and Alexa Fluor 488 anti-mouse IgG, Invitrogen) at room temperature for 1 hr. Then, blastocysts were counter-stained with 10 μg/ml Hoechst 33342 at room temperature for 20 min. Stained blastocysts were mounted on a glass slide containing ProLong Gold antifade solution (Invitrogen), covered by a coverslip, and imaged using an inverted fluorescence microscope.

Cells were fixed in 4% paraformaldehyde for 10 minutes, washed with PBS and permeabilized in 0.1% triton in PBS for 10 minutes. Cells were then blocked for 1 hour in 2% serum, 3% BSA and 0.1% triton in PBS and then primary antibody was added in the same solution overnight at 4°C. Cells were washed in PBS for 3 10 minute washes and put in secondary antibody for 1 hour at room temperature. After an additional 3 washes, coverslips were mounted with prolong gold antifade solution (Invitrogen). For surface staining assays, GluA1 antibody was added for 30 minutes to the media live cells at 37°C before fixation. Cells were then blocked without triton and secondary antibody was added immediately following the blocking step. Antibodies used were Brd4 (BethylA301-985A, 1:1000), Arc (Santa Cruz 365736, 1:100), GluA1 (Millipore 2263, 1:300), CK2 (Peirce/Thermo PA5-28686, 1:100), H4K16ac (Abcam 109463, 1:500) and secondary antibodies were AlexaFlour 647 Donkey anti-mouse (Jackson 715-605-150, 1:500) and Rhodamine Red-X goat anti-rabbit (Invitrogen R6394, 1:500).

After fluorescence imaging of the organs, they were embedded in O.C.T and sectioned at 5 μm thickness, and mounted on to glass slides. The slides were fixed and permeabilized with cold acetone, followed by blocking with 1% BSA for 1 hour. DAPI or EDB-FN specific antibody, BC-1 (Abcam, Cambridge, MA), were applied subsequently for staining. Unbound antibodies were washed with TBS-T (0.1%). Alexa Fluor 594-conjugated goat anti-mouse IgG H&L (Abcam) was used as the secondary antibody. Prolong Gold anti-fade solution (Invitrogen, Grand Island, NY), was used to cover the slides. The stained tissue sections were imaged using confocal laser scanning microscopy.

4T1 cells were seeded (1.5 × 10⁵ cells/well) in 6 well plates containing glass coverslips and incubated for 6 hrs under normoxia or hypoxia. Cells were treated with 33.3 μg mL^‒1 of Cat-Cy5-IONP or the molar equivalence of catalase-Cy5 for 16 hrs. The cells were washed thrice with cold PBS and fixed with 4% formaldehyde for 15 min and stained with and WGA-555 plasma membrane stain (Invitrogen, Carlsbad, CA). Coverslips were mounted on microscope slides using Prolong Gold Antifade solution (Invitrogen, Carlsbad, CA) containing DAPI nucleus stain. Images were acquired on a LSM 510 Meta confocal fluorescence microscope (Carl Zeiss, Inc., Peabody, MA) with appropriate filters.