Cells transfected with the Phox2B constructs were fixed in 3% (wt/vol) paraformaldehyde/phosphate buffered saline (PBS) for 10 min followed by permeablization using 0.4% (wt/vol) Triton X-100/PBS for 15 min. Expressed Phox2B was labeled with rabbit polyclonal anti-myc (ab9106; Abcam) followed by a secondary antibody incubation using Alexa-Fluor 488 conjugated goat anti-rabbit (Life Technologies). DNA was detected with Hoechst dye 33258. Coverslips were mounted using 10% (wt/vol) Mowiol 4–88 (Polysciences). Images were obtained using Nikon Ni-E microscope (40×/0.75 Plan Fluor; Nikon) with a CCD camera (CoolSNAP Myo; Photometrics) linked to a workstation running NES-Element software (Nikon) was used.
Mowiol 4 88
Manufactured by Polysciences
Sourced in United States
Mowiol 4–88 is a polyvinyl alcohol (PVA) product commonly used in laboratory settings. It is a water-soluble polymer with a molecular weight range of 31,000 to 50,000 g/mol and a degree of hydrolysis between 86% and 89%. Mowiol 4–88 is typically used as a binder, film-forming agent, and thickening agent in various applications.
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Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (6 mentions)
Eclipse ni e, by Nikon (5 mentions)
Nis elements software, by Nikon (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Dabco, by Merck Group (3 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (3 mentions)
Cm3050s cryostat, by Leica (3 mentions)
Alexa fluor 488 conjugated streptavidin, by Thermo Fisher Scientific (3 mentions)
A11004, by Thermo Fisher Scientific (3 mentions)
710 confocal microscope, by Zeiss (3 mentions)
Ab13970, by BD (2 mentions)
Ab9361, by Merck Group (2 mentions)
Cy5 nhs ester, by GE Healthcare (2 mentions)
P 30 gel exclusion column, by Bio-Rad (2 mentions)
Dylight 488, by Jackson ImmunoResearch (2 mentions)
Ab112, by Abcam (2 mentions)
Alexa fluor 568 conjugated goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Ab129184, by Abcam (2 mentions)
A 21235, by Thermo Fisher Scientific (2 mentions)
29 protocols using mowiol 4 88
1
Immunofluorescent Labeling of Phox2B
2
Fluorescence Imaging of Protein Localization
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The cells were plated on cover glass (thickness No 1.5) for 24 hr, followed by Flag-ASNA1 plasmid transfection or Flag-FAF1 tetracycline induction for 24 hr, and treated with MG132 for 2 hr where indicated. Cells were then fixed in 3.7% paraformaldehyde/PBS at room temperature for 15 min, permeabilized in 0.2% Triton X-100/PBS for 5 min, and blocked in 3% FBS in PBS/0.05% Tween for 45 min. Antibodies diluted in 3% FBS in PBS/0.05% Tween were sequentially added to the cells and incubated for 1 hr at room temperature followed by three washes with PBS/0.05% Tween. Cells were then incubated for 5 min with 4',6-diamidino-2-phenylindole (DAPI). Finally, the samples were washed three times with PBS/0.05% Tween, three times with PBS, and twice with water before mounting on microscope slides with Mowiol 4-88 (Polysciences, Eppelheim, Germany). Images were obtained with a DeltaVision Spectris microscope (Applied Precision, Issaquah, USA), using a CoolSNAP HQ camera (Roper Scientific, Martinsried, Germany) and a 100× 1.4 NA objective (Olympus, Southend-on-Sea, UK). The SoftWorx software (Applied Precision) was used for image acquisition and deconvolution.
3
Multicolor Immunofluorescence Imaging Protocol
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Tissue was fixed in 4% PFA/PBS at room temperature for 10 minutes and equilibrated in 30% sucrose overnight at 4°C. Tissue was embedded in O.C.T. (TissueTek) and stored at −80°C. Sections were cut at 10 µm using a Leica CM3050S cryostat and incubated with primary antibodies overnight at 4°C. Primary antibodies were rabbit anti-TH (Abcam, ab112), chicken anti-GFP (Abcam, ab13970), rat anti-CD31 (BD Pharmingen, 553370), chicken anti-β-galactosidase (Abcam, ab9361), and mouse anti-smooth muscle actin (Sigma, A5228) used at 1:500. Mouse anti-smooth muscle actin antibody was directly conjugated to Cy5 NHS ester (GE Healthcare), and unbound dye was removed on a P-30 gel exclusion column (BioRad)35 . Incubation with secondary antibodies was 45 minutes at room temperature. Secondary antibodies were conjugated to either Alexa Fluor 488, Alexa Fluor 555 (Life Technologies), or DyLight 488 (Jackson ImmunoResearch). Staining with 4’,6-diamidino-2-phenylindole (DAPI; 1 ng/ml, Life Technologies) was performed after incubation with secondary antibodies for 5 minutes at room temperature. Sections were mounted in Mowiol 4–88 (Polysciences) with DABCO (25 mg/ml, Sigma-Aldrich) and visualized on a Zeiss Axiophot fluorescence microscope. Tissue samples from three or more animals were stained for representative data shown (Fig. 1g, h , Fig. 3a, b , and Extended Data Fig. 3a–c, f–h ).
4
Immunofluorescence Staining Protocol
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Cells grown on glass coverslips were fixed in 3% (wt/vol) paraformaldehyde/phosphate-buffered saline (PBS) for 10 min and permeabilized by 0.4% (wt/vol) Triton X-100/PBS for 15 min. For labeling fusion protein purposes, a mouse anti-hemagglutinin primary antibody was used (HA; 1:1000; 12CA5; Covance, Princeton, NJ, USA). The primary antibody was detected using Alexa Fluor 488–conjugated goat anti-mouse (1:1000; A11029; Thermo Fisher Scientific, Waltham, MA, USA) or Alexa Fluor 568–conjugated goat anti-mouse (1:1000; A11004; Thermo Fisher Scientific, Waltham, MA, USA). Alexa Fluor 488–conjugated streptavidin (S32354; Thermo Fisher Scientific, Waltham, MA, USA) or Alexa Fluor 568-conjugated streptavidin (S11226; Thermo Fisher Scientific, Waltham, MA, USA) was used to detect biotinylated proteins. DNA was detected with Hoechst dye 33342. Coverslips were mounted using 10% (wt/vol) Mowiol 4-88 (Polysciences, Inc., Warrington, PA, USA). Confocal images were obtained using a Nikon A1 confocal microscope (60 ×/1.49 oil APO TIRF Nikon objective) with a charge-coupled device camera (CoolSnap HQ; Photometrics, Tucson, AZ, USA) linked to a workstation running NIS-Elements software (Nikon, Melville, NY, USA). Epifluorescence images were captured using a Nikon Eclipse NiE (20 ×/0.75 Plan Apo Nikon objective) microscope.
5
Immunofluorescence Microscopy of Toxoplasma-Infected Cells
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Cells were plated on coverslips (12mm, ♯1.5, ThermoFisher) (coated with 1% gelatin) in a 24-well plate and cultured, IFNγ–stimulated and infected with Toxoplasma as described above. The cells mounted on coverslips were prepared for imaging as detailed in S1 Appendix . Primary and secondary antibody incubations were carried out sequentially followed by washes of 3x 1ml PGAS, 2x 1ml PBS, and then by 1ml PBS containing 1μg/ml Hoechst 33342 (Life Technologies). Finally samples were washed twice in dH2O prior to mounting on glass slides with Mowiol 4–88 (Polysciences Inc.). Staining protocol is detailed further in S1 Appendix . Slides were viewed on a Zeiss Axioplan II Epifluorescence microscope using x100 objective, imaged with an AxioCam HRC camera and analysed with Axiovision 4.8 software or on an SP5-invert Confocal microscope using x100 objective and analysed using LAS-AF software.
6
Immunofluorescence Staining of Cell-Cell Junctions
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First, cells were fixed for with 4% PFA diluted in PBS. Next, the cell membrane was permeabilized with 0.5% Triton X-100 for . Cells were then washed twice with TBS and blocked at room temperature for with a blocking buffer solution containing TBS, 1% bovine serum albumin (BSA, Sigma-Aldrich), and glycine (Sigma-Aldrich). Then, cells were incubated for in a dilution of primary antibodies with blocking buffer. For E-cadherin stainings, a 1:200 dilution of DECMA-1 (Thermo Fisher 14-3249-82) was used and for vinculin stainings a 1:400 dilution of hVIN-1 (Sigma-Aldrich V9131) was used. Cells were then washed three times with TBS for each. Then cells were incubated in a dilution of secondary antibodies, Alexa 555-conjugated phalloidin and DAPI in blocking buffer. For E-cadherin stainings, a 1:1000 dilution of Alexa 647-conjugated anti-rat (Sigma-Aldrich SAB4600186) was used; for vinculin stainings, a 1:1000 dilution of Alexa 647-conjugated anti-mouse (Thermo Fisher A-21235) and a 1:1000 dilution for phalloidin and DAPI. Fixed cells were then mounted with Mowiol 4-88 (Polysciences, Inc) onto glass slides and kept at °C until imaging.
7
Immunofluorescence Assay for BAF, pBAF, and HSP90
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Cells were grown on glass coverslips and fixed in 3% (wt/vol) PFA/PBS for 10 min and permeabilized by 0.4% (wt/vol) Triton X-100/PBS for 15 min. For immunofluorescence of HSP90, cells were fixed in 3% PFA for 10 min, then in −20°C methanol for 10 min, and permeabilized by 0.4% Triton X-100/PBS for 15 min. Cells were labeled for 1 h in primary antibodies: rabbit anti–BAF (1:100; ab129184; Abcam), rabbit anti-pBAF (specific for phosphorylated BAF; 1:200; generous gift from Robert Craigie, National Institutes of Health, Bethesda, MD), rabbit anti–LEMD2 (1:100; HPA017340; Atlas Antibodies), and mouse anti–HSP90 α/β (1:100; sc-13119; Santa Cruz Biotechnology). Primary antibodies were detected using Alexa Flour 488–conjugated goat anti–rabbit (1:1000; A21235; Thermo Fisher Scientific), Alexa Fluor 568–conjugated goat anti–mouse (1:1000; A11036; Thermo Fisher Scientific) and Hoescht dye 33342 to detect DNA. Coverslips were mounted using 10% (wt/vol) Mowiol 4-88 (Polysciences). Epifluorescent images were captured using a Nikon Eclipse NiE (40×/0.75 Plan Fluor Nikon objective; 20x/0.75 Plan Apo Nikon objective) microscope at room temperature with a charge-coupled device camera (CoolSnap HQ; Photometrics) linked to a workstation running NIS-Elements software (Nikon). All images were processed in Adobe Photoshop CS6 for cropping and brightness/contrast adjustment when applicable.
8
Immunofluorescence of Nuclear Proteins
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Cells were grown on glass coverslips and fixed in 3% (wt/vol) paraformaldehyde/phosphate buffered saline (PFA) for 10 min and permeabilized by 0.4% (wt/vol) Triton X-100/PBS (PBST) for 15 min. Cells were labeled for 1 hr with rabbit anti-BAF (1:100; ab129184; Abcam), rabbit anti-Emerin (1:100; ab156871; Abcam) or rabbit anti-phospho-Lamin A/C (pSer22; 1:100; 2026S; Cell Signaling). Primary antibodies were detected using Alexa-Flour 488 conjugated goat anti-rabbit (1:1000, A21235, Thermofisher) and Hoescht dye 33342 to detect DNA. Coverslips were mounted using 10% (wt/vol) Mowiol 4–88 (Polysciences). Epifluorescent images were captured using a Nikon Eclipse NiE (40×/0.75 Plan Fluor Nikon objective; 40x/0.75 Plan Apo Nikon objective) microscope at room temperature with a CCD camera (CoolSnap HQ; Photometrics) linked to a workstation running NIS-Elements software (Nikon Melville, NY). All images were processed in Adobe Photoshop CS6 for cropping and brightness/contrast adjustment where applicable.
9
Immunohistochemical Analysis of VCP and Methylation
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The various tissues were fixed in 10% neutral buffered formalin and afterwards embedded in paraffin. For immunohistochemistry, tissue sections (4 μm thickness) were deparaffinized, rehydrated and subjected to heat-induced antigen retrieval in Tris-EDTA buffer (10mM Tris, 1mM EDTA, pH 9). The sections were permeabilized with 0.5% Tween 20 in PBS for 15 min at room temperature and blocked with 5% BSA, 5% goat serum in PBST (0.05% Tween) for 90 minutes at room temperature. Slides were then incubated with the primary antibodies overnight at 4°C. After 3 washes with PBST for 5 min, the sections were incubated in the dark with the secondary antibodies. For counterstain DAPI (1 μg/ml) was used and Mowiol 4–88 (Polysciences) was used for mounting. Primary antibodies: mouse anti-VCP (1:250, Abcam, ab11433), rabbit polyclonal anti-K315me3 (1:500, New England Peptides, custom antibody against synthetic peptide H2N-AIAPKRE(3me)KTHGEVERR-OH, double affinity purified), anti-VCPKMT (1:500, serum of rabbits immunized with recombinant human VCPKMT [6 (link)]). Secondary antibodies: goat anti-rabbit-Alexa 488 (1:500, Life Technologies), goat anti-mouse-Alexa 594 (1:500, Life Technologies). Images of fluorescently stained sections were acquired using AxioCam MRRev3 camera on an Axio Observer. Z1 microscope (Carl Zeiss).
10
Immunohistochemical Analysis of Brain Tissues
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Sections were selected for histological examination based on MRI and DTI findings. After antigen epitope retrieval with 1X Citrate buffer (Thermo Scientific, Waltham, Mass) for 20 min in a 60°C water bath, the sections were rinsed with PBS and incubated in blocking buffer solution (0.1% Triton X-100, 3% normal goat serum in PBS) for 2 h at room temperature (RT), followed by an overnight incubation with primary antibodies at 4°C. Primary antibodies used included markers for astrocytes (GFAP, 1:500, Abcam, ab4674); macrophage/microglia (Iba1, 1:1,000, Wako, 019019741); myelin oligodendrocyte glycoprotein (MOG, 1:300, Sigma, SAB1406138); and dendritic microtubule-associated protein 2 (MAP2, 1:200, Abcam, ab5392). After three PBS washes, staining was revealed by 2-h incubation at RT with the appropriate Alexa 488 or 555 conjugated secondary antibodies (Invitrogen) diluted 1:500 in blocking buffer. DAPI (4′,6-Diamidino-2-phenylindole, 1:2,000, Invitrogen, D21490) was added to the secondary antibody solution for nuclear counterstaining. Following three more washes in PBS, the sections were mounted onto slides and coverslipped with Mowiol 4-88 (Polysciences) mounting medium. Immunolabeled brain sections were then analyzed and imaged using a Zeiss Axiovert 200 microscope equipped with an Apotome and Axiovision 4.7 Zen software (Carl Zeiss, Inc, Oberkochen, Germany).
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