Prolong gold mounting medium with dapi

For immunofluorescent staining of brain tissue, mice were perfused with 4% paraformaldehyde and post fixed for an hour before transferring into successive sucrose solutions at 15% and 30%. 20 μm thick cryosections were prepared for immunohistochemistry (IHC). Tissue was incubated with 1.5% normal goat serum (NGS) (Life Technologies) and 0.1% Triton X-100 (Sigma) in PBS for 60 min at room temperature. Sections were incubated with primary antibodies (Rat Anti-Somatostatin 1:250 [Millipore MAB354]; Anti-parvalbumin 1:1000 [Sigma P3088]; Anti-VIP 1:250 [ImmunoStar 20077]; Anti-MeCP2 1:250 [Millipore 07–013]) in the blocking buffer overnight at 4°C. After washing three times with buffer, sections were incubated with secondary antibodies for 1 hr at room temperature (secondary antibodies: Alexa Fluor 488, 594 or 647 [Life Technologies, 1:1000]). Finally, coverslips were mounted using ProLong Gold Mounting Medium with DAPI (Life Technologies) and imaged at 10x. Quantifications were performed in Adobe Photoshop. Pictures were divided into a grid measuring 1 × 1 mm in total and cells were counted in each grid square. The number of cells positive for antibody staining against MeCP2 was counted to assay the proportion of co-expressing cells.

One day prior transfection and/or electroporation cells were seeded on glass coverslips in a 35 mm² dish. For antibody staining cells were fixed with 4% PFA and washed three times with PBS. Coverslips were blocked for 20 min with PBS containing 2% FBS, 1% BSA and 0.4% Triton-X100. After incubation with primary antibodies anti-HA (H3663, Sigma-Aldrich, 1:200) and anti-ubiquitin P4D1 (Santa Cruz, 1:100, sc-8017) for two hours, coverslips were washed three times with PBS and incubated with secondary antibody (anti-mouse-Alexa488, Jackson Laboratories) for one hour. Lastly, coverslips were washed three times with PBS and mounted on object glasses using ProlongGold mounting medium with DAPI (Life technologies, P36931). All procedures were performed at room temperature.

For immunofluorescence analysis, cells were seeded in 8-well chamber slides (LabTek Chamber slides, Thermo Fisher Scientific), cultured and treated as described in the previous section. At the end of treatments cells were fixed with 4% Formaldehyde and, for intracellular antigens detection, permeabilized with Triton X-100 0.5% in PBS. Blocking with BSA 3% was performed to prevent non-specific binding of the antibodies. Cells were then incubated with primary antibody diluted in BSA 3% for 1 hour and for 30 minutes with appropriate secondary antibodies. Coverslips were mounted on slides using ProLong Gold mounting medium with DAPI (Life Technologies). Slides were imaged with a Zeiss Axioskope 2 microscope (Zeiss) equipped with fluorescence lamp and filters and a high-resolution digital camera (C4742–95, Hamamatsu Photonics). Single channel grey-scale images were quantified using ImageJ Software [46 (link)]. Threshold was fixed and applied to all images stained with the same antibody. For nuclear antigens, images were processed and threshold was fixed in order to measure only nuclear staining signal. Obtained fluorescence intensity measurements were normalized to DAPI fluorescence signal. The images were then processed with Adobe Photoshop CS6 software for color assignation to the corresponding fluorescence signal.

Cells (SKOV3) were cultured on glass coverslips for 24 h. Cytoplasmic mRNP granule formation was triggered by treatment with 5 μg/ml sodium arsenite (Sigma-Aldrich) for one hour. Cells were washed before being incubated with PHEM fixative (4% PFA, 60 mM PIPEs, 25 mM HEPES, 10 mM EGTA, 4 mM MgCl₂) for 10 min. Fixative was removed and cells were washed and blocked in PBSTB buffer (1% BSA, 0.1% TritonX-100) for 1 h. Cells were stained with antibodies to DCP1A (Abnova), PABP (Abcam) and LARP1 (SDIX- Novus Biologicals). Primary antibody solution was applied and incubated overnight at 4°C. After washing, Alexa Fluor-conjugated secondary antibodies (Life Technologies) were applied and incubated at room temperature for 1 h. Cells were washed and mounted with ProLong Gold mounting medium with DAPI (Life Technologies). Immunofluorescence staining was analysed using Leica 500 confocal microscope and images processed with Leica LAS AF lite software.

Eyes were fixed in 4% PFA for 2 h at room temperature and equilibrated in 30% sucrose at 4 °C, followed by embedding in OCT. Sections (10-μm thick) were heated at 98 °C for 10 min in citric acid buffer for antigen retrieval, blocked with 10% goat serum for 1 hour, and incubated with mouse HIF-1α (1:100, BD Biosciences, 610958), rabbit Ki-67 (1:200, RM-9106, Thermo Scientific), rabbit PFKFB3 (1:100, Proteintech, 13763-1-AP), rat CD31 (1:25, Invitrogen, DIA-310) and/ or Alexa-594 labeled Griffonia simplicifolia isolectin B4 (1:100, Invitrogen, Cat. No. 121413) overnight at 4 °C, followed by incubation with fluorescence-conjugated secondary antibody (1:250, Molecular Probes, Life Technologies, Carlsbad, CA,USA) for 1 hour. For Ki-67/ ERG double immunofluorescent staining, sections were then stained with Anti-ERG antibody (Alexa Fluor® 594) (1:200, Abcam, Clone number: EPR3864) overnight at 4 °C. Sections were washed with PBS, immersed in ProLong Gold mounting medium with DAPI (Invitrogen) to visualize the nuclei, and examined using confocal microscopy. For all immunofluorescence experiments, parallel groups of sections were stained with only primary or secondary antibody as negative controls.

Immunohistochemical analysis was performed as previously published (Duong et al. 2018). For immunolocalization of antigens, RGCs plated on 14 mm coverslips were fixed in 4% paraformaldehyde followed by permeabilization using 0.1% Triton X-100 for 5 min. Cells were incubated for 1 h at RT with a blocking buffer (1% BSA, 5% normal goat serum, 0.1% Triton X-100 in 1% PBS). RGCs were incubated with primary antibodies diluted in 1% BSA, 0.1% Triton X-100 in 1% PBS buffer for overnight at 4 °C. After washing thrice with 1X PBS, cells were incubated with secondary antibodies diluted in 1% BSA, 0.1% Triton X-100 in 1% PBS buffer for 1 h at RT. The coverslips were then mounted on slides using prolong Gold mounting medium with DAPI (4′,6-diamidino-2-phenylindole) (Invitrogen). RGCs immunohistochemistry was performed as described earlier^{36 (link),38 (link)–40 (link)} using SOX2, PAX6, RAX, TUJ1, Thy1/CD90, MAP2, GFAP, CRALBP, BRN3A, and RBPMS antibodies with dilution as provided in Supplementary Table S1. Slides were observed under an Olympus FV1000 Confocal microscope and images were captured with the use of appropriate filters and lasers.