Cells seeded on chamber slide were incubated in Fixation buffer (Biolegend, UK) for 10 min at room temperature, washed three times with PBS, permeabilised (0.5% Triton X-100 in PBS) for 10 min and treated with blocking buffer (PBS 1% BSA or 10% normal serum from the species in which the secondary was raised) prior to incubation with primary antibodies overnight at 4°C. The following primary antibodies were used: anti-vinculin (V9264, Sigma, UK) and Alexa Fluor™ 488 Phalloidin (A12379, Invitrogen, UK). Samples were then incubated with donkey anti-mouse Alexa fluor 647 (A31571, Invitrogen, UK) for 1h at RT. Slides were mounted with SlowFade™ Diamond Antifade Mountant with DAPI (S36968, Invitrogen, UK) to counterstain nuclei. Samples were visualized using an EVOS (EVOS™ FL Auto 2, Thermofisher, UK) microscope.
Slowfade diamond antifade mountant with dapi
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Japan
SlowFade Diamond Antifade Mountant with DAPI is a ready-to-use aqueous mounting medium designed to retard photobleaching of fluorescent dyes, including DAPI. It is formulated to maintain the integrity and fluorescence of labeled samples.
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Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (6 mentions)
Biocoat poly d lysine 4 well culture slides, by Corning (4 mentions)
Bz x700, by Keyence (4 mentions)
Blocking one, by Nacalai Tesque (4 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Celltracker cm dii dye, by Thermo Fisher Scientific (2 mentions)
Millicell ez slide, by Merck Group (2 mentions)
Goat serum, by Fujifilm (2 mentions)
Fugene hd transfection reagent, by Promega (2 mentions)
Biorevo bz 9000 fluorescence microscope, by Keyence (2 mentions)
Picrosirius red fast green, by Chondrex (2 mentions)
At2 slide scanner, by Olympus (2 mentions)
Fluorescence upright microscope at 20x, by Olympus (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Nacalai Tesque (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Anti ssea4, by Cell Signaling Technology (2 mentions)
53 protocols using slowfade diamond antifade mountant with dapi
1
Immunofluorescence Staining of Cytoskeletal Proteins
2
Immunofluorescent Localization of DR2a and DR2b
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BLS-DR2a or BLS-DR2b cells were plated onto poly-L-Lysine (Sigma-Aldrich) -coated slides. The fixation, permeabilization, and blocking of nonspecific binding sites of the cells were performed using the Image-iT® Fix-Perm Kit (Invitrogen, California, USA) according to the manufacturer’s instructions. The cells were then incubated with 10 μg/ml primary anti-DR2a or anti-DR2b antibody overnight at 4°C. Following several wash steps with PBS + 0.5% BSA, the cells were incubated with 1 μg/ml Alexa Fluor 488-conjugated goat anti-mouse IgG1 secondary antibody (Invitrogen) to target anti-DR2a antibody or 10 μg/ml Alexa Fluor 647-conjugated goat anti-mouse IgG2b secondary antibody (Invitrogen) to target anti-DR2b antibody for 1 h at room temperature. Thereafter, cells were washed several times with PBS + 0.5% BSA and mounted with SlowFade Diamond Antifade Mountant with DAPI (Invitrogen) and coverslips. The images were captured using fluorescence microscopy (Zeiss, Oberkochen, Germany) and analyzed using ImageJ (NIH).
3
Immunostaining of Stem Cell Derivatives
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Generated iPSCs were plated on Matrigel (Corning, NY, USA)-coated cover slips and grown in Essential 8 medium. Then, the cells were fixed in 4% PFA, permeabilized with 0.5% Tween and blocked in 1% bovine serum albumin. Next, the cells were incubated overnight with the primary antibodies (listed in Table S7 ) at 4 °C. Thereafter, the cells were washed 3× with PBS for at least 5 min each time and incubated 1 h with fluorescent-dye conjugated secondary antibodies at room temperature and again washed 3× with PBS for at least 5 min. SlowFade Diamond Antifade Mountant with DAPI (Invitrogen) was used for nuclear staining. Images were captured on a Leica SP5 confocal microscope.
4
Visualizing pGFPns Delivery to HSPCs
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To visualize the delivery of pGFPns to HSPCs, at 24 and 72 hours, HSPCs from the coculture with Cy5-pGFPns were examined by ELYRA PS.1 Superresolution Microscopy (Zeiss). Briefly, cells were first stained with CellTracker CM-DiI Dye (Invitrogen) for lipid membrane staining and seeded onto poly-l -lysine–coated coverslips. After 10 min, cells were fixed with 4% paraformaldehyde for 10 min at room temperature. After washing with PBS thrice, samples were mounted with SlowFade Diamond Antifade Mountant with DAPI (Invitrogen) for imaging.
5
Lysosomal Profiling in HaCaT Cells
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To assess the lysosomal number and distribution via LAMP1 staining, proinflammatory cytokine-stimulated HaCaT cells were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature (RT) and subsequently permeabilized with 0.1% Triton X-100 for 15 min at RT. Cells were then blocked with 3% BSA in PBS + 0.1% Tween for 1 h at 4 °C. Immunostaining was performed by overnight incubation with a LAMP1 primary antibody (#9091, Cell Signaling, Leiden, Netherlands; 1:200) in blocking solution at 4 °C. The following day, cells were washed three times with PBS and incubated with a secondary antibody (anti-rabbit IgG (H + L), F(ab′)2 fragment, Alexa Fluor® 594 Conjugate; #8889, Cell Signaling, Leiden, Netherlands; 1:1000) in blocking solution for 1 h at 4 °C. After three washes with PBS, coverslips were mounted in SlowFade™ Diamond Antifade Mountant with DAPI (Invitrogen, Thermo Fisher Scientific). Each treatment was analyzed in triplicate in three independent experiments.
6
Immunohistochemical Analysis of Dental Tissue
7
Immunostaining of Prostate Tumor Tissue Sections
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Prostate tumor samples isolated from mice were fixed overnight at 4 °C using Zamboni’s fixative (2% paraformaldehyde and 0.5% picric acid in 1× PBS). Then, the samples were washed thrice in PBS and cryoprotected using 20% sucrose overnight at 4 °C. After three PBS washes, the tissues were embedded in Tissue-Plus™ O.C.T. Compound. Twelve µm thick sections were then taken on slides and the sections were blocked using 5% donkey serum containing 0.3% Triton X-100 for 30 min. The sections were afterward incubated for 1 h at room temperature (RT) with one of the following primary antibodies: mouse anti-Chromogranin A Clone DAK-A3 (1:100, Agilent DAKO M0869, Santa Clara, CA, USA), rabbit anti-Chromogranin B (1:100, Invitrogen PA5-52605, Waltham, MA, USA), or rabbit anti-Synaptophysin (D8F6H) XP® (1:100, Cell Signaling 36406, Danvers, MA, USA). Afterward, the sections were incubated with either goat anti-mouse Alexa Fluor 546 (Invitrogen A21045) or goat anti-rabbit Alexa Fluor 546 (Invitrogen A11035) for 1 h in the dark at RT. The sections were mounted using SlowFade™ Diamond Antifade Mountant with DAPI (Invitrogen S36973), and all images were captured using an Axio Observer 7 inverted fluorescence microscope (Carl Zeiss, Oberkochen, Germany).
8
Immunofluorescence Staining of TDP-43 in SH-SY5Y Cells
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SH-SY5Y cells expressing TDP-43 WT or mNLS were cultured in 8-well chamber slides (Millicell EZ slides, Millipore), treated with DMSO or 10 μM etoposide (Sigma, #341205) for 2 h following a recovery step at pre-defined timepoints, xed in 4% PFA in phosphate-buffered saline (PBS) for 20 min and permeabilized with 0.5% Triton X-100 in PBS for 20 min at room temperature. Blocking was performed with 3% BSA solution in PBS for 30 min. Then cells were incubated with primary antibodies overnight at 4°C in 1% BSA solution and uorescent secondary antibodies for 1 h at 37°C. Slides were washed 3 times and counterstained with DAPI.
For IF on tissue samples, sections were blocked in 10% normal goat and/or donkey serum, 1% gelatin in 1x TBS-T for 30 min prior to primary antibody incubation. Alexa Fluor 488 and 568 conjugated antibodies raised in goat or donkey were used as secondaries (1:500, Molecular Probes, Invitrogen). Nuclei were counterstained with SlowFade™ Diamond Antifade Mountant with DAPI (Invitrogen, #S36964). Images were captured in AXIO Observer inverted fluorescence microscope (Carl Zeiss).
For IF on tissue samples, sections were blocked in 10% normal goat and/or donkey serum, 1% gelatin in 1x TBS-T for 30 min prior to primary antibody incubation. Alexa Fluor 488 and 568 conjugated antibodies raised in goat or donkey were used as secondaries (1:500, Molecular Probes, Invitrogen). Nuclei were counterstained with SlowFade™ Diamond Antifade Mountant with DAPI (Invitrogen, #S36964). Images were captured in AXIO Observer inverted fluorescence microscope (Carl Zeiss).
9
Immunostaining of pGCTB-SCs on Poly-L-Lysine
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pGCTB-SCs were seeded on poly-L-lysine (Fujifilm Wako)–coated cover glass at a density of 10 × 104 cells and cultured for 48 h. After the cells were treated with each reagent for the indicated periods, they were fixed with 4% paraformaldehyde (FUJIFILM Wako) for 10 min at RT, permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 15 min, and blocked with 10% goat serum (FUJIFILM Wako) for 30 min. Subsequently, the cells were incubated at 4 °C overnight with a mixture of primary antibodies diluted in 1:200 in Can Get Signal Immunostain Solution A (TOYOBO, Osaka, Japan). Samples were then washed three times with PBS and incubated with Alexa Fluor® 488 and 594 diluted in 1:200 for 1 h at RT. SlowFade Diamond antifade mountant with DAPI (Invitrogen) was used as a mounting solution. Immunostaining was visualized using fluorescence microscopy (BZ-X800; Keyence).
10
Evaluating TFEB Nuclear Translocation in Cytokine-Stimulated HaCaT Cells
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To analyze the proinflammatory effect on TFEB nuclear translocation, cytokine-stimulated HaCaT cells were fixed with 4% PFA for 10 min at RT. For endogenous TFEB staining, cells were permeabilized with 0.1% Triton X-100 for 15 min at RT, followed by blocking with 3% BSA in PBS + 0.1% Tween for 1 h at 4 °C. Immunostaining was performed by overnight incubation with a TFEB primary antibody (#4240, Cell Signaling, Leiden, Netherlands; 1:1000) in blocking solution at 4 °C. The following day, cells were washed three times with PBS, and incubated for 1 h with a goat anti-rabbit IgG Alexa Fluor 488 secondary antibody (#A-11034, Invitrogen, Thermo Fisher Scientific,; 1:500) in blocking solution at 4 °C. Following an additional three washes with PBS, coverslips were mounted in SlowFade™ Diamond Antifade Mountant with DAPI (Invitrogen, Thermo Fisher Scientific). Each treatment was analyzed in triplicate in three independent experiments. An additional control with 0.3 µM Torin 1 (MTORC1 inhibitor) was applied for the last 3 h of treatment.
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