Cells were seeded on sterile coverslips placed in 6-well plates. After cells were infected with S. lutetiensis for 2 h, they were fixed in 4% paraformaldehyde for 10 min and then permeabilized with 0.2% Triton X-114 in PBS for 15 min. After being washed with PBS, cells were blocked with 3% BSA for 1 h at RT. Slides were incubated with anti-α-tubulin antibody (1 : 200 diluted in PBS) overnight at 4°C and then incubated with peroxidase-conjugated AffiniPure (diluted 1 : 100 in PBS) second antibody for 1 h at 37°C, before nuclei and intracellular bacteria were stained with DAPI. Between steps, cells were thrice-washed with PBS. Ultimately, all slides were mounted with antifade mounting medium and examined on a Nikon A1HD25 confocal microscope with a 100x oil immersion objective. Imaging used laser wavelengths of 488 and 561 nm.
A1 hd25 confocal microscope
Manufactured by Nikon
Sourced in Japan
The Nikon A1 HD25 is a high-performance confocal microscope. It features a 25 mm field of view, high-resolution imaging, and advanced optics for detailed biological and materials research.
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Lab products found in correlation
Alexa fluor 488 donkey anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 donkey anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Ab227883, by Abcam (1 mentions)
P0102, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Nis elements viewer, by Nikon (1 mentions)
M csf, by Sino Biological (1 mentions)
Tunel assay kit, by Thermo Fisher Scientific (1 mentions)
Eclipse ts2r, by Nikon (1 mentions)
Mca2336, by Abcam (1 mentions)
Anti α tubulin, by Cell Signaling Technology (1 mentions)
Cryostat, by Leica (1 mentions)
Mitotracker green, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit af488, by Abcam (1 mentions)
Vectashield antifade reagent with dapi, by Vector Laboratories (1 mentions)
Triton x 100, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Normal goat serum (ngs), by OriGene (1 mentions)
549 conjugated secondary antibodies, by EarthOx (1 mentions)
15 protocols using a1 hd25 confocal microscope
1
Intracellular Bacteria Immunohistochemistry
2
Immunofluorescence Analysis of METTL3, DNMT3A, and DNMT3B
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C2C12 cells were fixed with 4% paraformaldehyde for 15 min at room temperature. After permeabilization with 0.25% Triton X-100 for 10 min, the cells were blocked by using 1% bovine serum albumin for 30 min. Then, the cells were incubated overnight with Anti-METTL3 antibody (67733–1-Ig; Protentech, Wuhan, China), and Anti-DNMT3A antibody (ab228691; Abcam, Cambridge, England),or Anti-DNMT3B antibody (ab227883; Abcam, Cambridge, England) diluted in PBST containing 1% bovine serum albumin at 4 °C. The cells were incubated with Alexa Fluor 647 donkey anti-mouse IgG (H + L), (A-31571; Invitrogen, MA, USA) or Alexa Fluor 488 donkey anti-rabbit IgG (H + L) (A-21206; Invitrogen, MA, USA) for 1 h at room temperature. Nuclei were stained with DAPI for 1 min. The cells observed microscopically using an A1HD25 confocal microscope (Nikon Instruments, Tokyo, Japan).
3
Imaging Analysis of Mitochondrial Proteins and Amyloid in Prion-Treated Cells
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N2a cells were cultured on 24-well coverslips and subsequently transfected with Mito-GFP, DsRed-Mito, Thioflavin T, or siRNA before treatment with PrP106−126 or the control peptide. The specific processes were carried out in accordance with our previous studies (Song et al., 2022 (link)). Cells were washed twice with PBS before being fixed with 4% paraformaldehyde for 30 min. After washing twice with PBS, the cells were treated with immunostaining permeabilization buffer containing X-100 (Beyotime biotechnology, P0096) for 5–10 min at room temperature to permeabilize them. The cells were then blocked using an immunostaining blocking buffer (Beyotime Biotechnology, P0102) for 1 h at room temperature, followed by overnight incubation with specific primary antibodies (as described in the immunoblotting section) at 4°C (West et al., 2015 (link)). Following the PBS rinsing step, the cells were incubated with secondary antibodies for 1 h at 37°C and then washed with PBS five times for 5 min each. The coverslips were mounted on microscope glass slides using a fluorescent antifading buffer (Bioworld Technology, BD5014). Images were acquired using a Nikon A1HD25 confocal microscope at a magnification of 100 × (oil immersion lens). Approximately 10–15 unique images were captured at random for each sample. The acquired images were quantified using ImageJ software.
4
Measuring Protein Dynamics via FRAP
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This experiment was done according to a previous study [30 (link)]. The cells that had stable β-actin-mCherry fusion protein expression were transferred to 35 mm glass-bottom cell culture dishes (Nest, China). Imaging was performed using the Nikon A1HD25 confocal microscope. In brief, three frames were imaged using low laser power and a rectangular region was subsequently bleached with 8 iterations at full laser power. Imaging was performed with 16 s time-lapse intervals for 180 s. Fluorescence recovery then followed with identical settings to prebleach/activation frames. Fluorescence intensity was measured from small areas of the bleached region. Recovery rate was calculated according to the formula: recovery rate = (Vt-Vpost)/(Vpre-Vpost), where Vt is the intensity at the indicated time point, Vpre is the intensity before bleaching, and Vpost is the intensity right after bleaching.
5
Immunofluorescence Imaging of Tight Junction Proteins
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Cells were seeded at 2.0e5 cells per 2.55 cm2 glass cover slip (Fisher Scientific, Waltham, MA). Cell layers were washed 3X in PBS. 2mL of 4% formalin were added to cover each cell layer for 3-minutes. The cell layer was once again washed three times with PBS and then blocked with 1% goat serum (Jackson ImmunoResearch, West Grove, PA). After blocking, preparations were exposed to rabbit anti-human occludin (1:300 dilution in 1% goat serum) and mouse anti-human claudin-4 (1:300 dilution in 1% goat serum) for 40-minutes (Fisher Scientific, Waltham, MA), washed and exposed to Alexa-flour 488 (green) goat anti-rabbit (1:400 dilution in 1% goat serum) and Cyanine3 (red) goat anti-mouse (1:400 dilution in 1% goat serum) for 40-minutes (Fisher Scientific, Waltham, MA). Finally, cell layers were exposed to DAPI (Fisher Scientific, Waltham, MA) for 1-minute, washed three times in PBS, and stored overnight at 4°C.
The following day, green and red fluorescence were observed via confocal laser scanning microscopy (Nikon A1 HD25 confocal microscope Melville, NY) and images were obtained using Nikon NIS Elements Viewer.
The following day, green and red fluorescence were observed via confocal laser scanning microscopy (Nikon A1 HD25 confocal microscope Melville, NY) and images were obtained using Nikon NIS Elements Viewer.
6
Osteogenic and Osteoclast Differentiation of Mouse Bone Marrow Stromal Cells
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Mouse bone marrow‐derived stromal cell (mBMSC) cultures were conducted as described previously.(28 ) For osteogenic differentiation, cultured mBMSCs were induced with osteogenic differentiation medium [α‐MEM containing ascorbic acid (50 μg/mL) and β‐glycerophosphate (10 mM) and dexamethasone (10−8 M)] for 20–25 days. The differentiation medium was then changed every 2 days. Osteoclast differentiation was performed as described previously.(28 ) In brief, bone marrow cells were induced to differentiate into osteoclasts with receptor activator of NF‐κB ligand (RANKL) (25 ng/mL) and macrophage colony‐stimulating factor (M‐CSF) (30 ng/mL) (Sino Biological, Beijing). The medium was refreshed daily for 7 days. Osteoclast formation was then examined under a Nikon A1 HD25 confocal microscope.
7
Comprehensive Immunofluorescence Analysis of iRPE and Mouse Retina
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iRPE cells were seeded on coverslips prior to microscopic examination. The gross morphology was determined using the light microscope Nikon ECLIPSE Ts2R. Fluorescence microscopy was carried out on 2% PFA‐fixed iRPE cells, using ZO‐1 (1:500, Cell signaling, #5406 S). Roche In Situ Cell Death Detection Kit, Fluorescein (Millipore Sigma, 11684795910) was used to detect cell death based on the provided protocol. The iRPE was subject to nuclear staining by Hoechst.
Retina and RPE were carefully dissected from the mouse eyes subject to DFO treatment. Untreated mice of similar ages were euthanized as the controls. The eyes were fixed by 4% PFA prior to immunofluorescence staining. The retina was stained with Arrestin 3 (1:400, Millipore Sigma, AB15282) for cone photoreceptors. The RPE was stained with phalloidin (1:1,000, Abcam, ab176756), ZO1 (1:150, Invitrogen, #61‐7300) and IBA1 (1:500, Fujifilm Wako, 019‐19741). Cell death was detected by the TUNEL assay kit (ThermoFisher Scientific, C10617) following the manufacturer's protocol with slight modifications, including extending the TdT reaction to overnight at 37°C. Both retina and RPE were counterstained with Hoechst.
All the immunofluorescence images were acquired with the Nikon A1 HD25 confocal microscope. The pictures were processed by ImageJ and GIMP 2.10.28.
Retina and RPE were carefully dissected from the mouse eyes subject to DFO treatment. Untreated mice of similar ages were euthanized as the controls. The eyes were fixed by 4% PFA prior to immunofluorescence staining. The retina was stained with Arrestin 3 (1:400, Millipore Sigma, AB15282) for cone photoreceptors. The RPE was stained with phalloidin (1:1,000, Abcam, ab176756), ZO1 (1:150, Invitrogen, #61‐7300) and IBA1 (1:500, Fujifilm Wako, 019‐19741). Cell death was detected by the TUNEL assay kit (ThermoFisher Scientific, C10617) following the manufacturer's protocol with slight modifications, including extending the TdT reaction to overnight at 37°C. Both retina and RPE were counterstained with Hoechst.
All the immunofluorescence images were acquired with the Nikon A1 HD25 confocal microscope. The pictures were processed by ImageJ and GIMP 2.10.28.
8
Immunostaining of Germ Cell Markers
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Follicles within the MCO were fixed by 4% PFA in DPBS for 30 min. Then the fixed cell was washed by 1 × TBST for 5 min twice. Then the sample was soaked in a blocking buffer composed of 1 × TBST with 10% donkey serum and 0.1% Triton-X for 1h. Preparing anti-Dazl (BIO-RAD, MCA2336) and anti-Amh (Abcam, ab272221) antibodies at 1 : 100 in blocking buffer and incubating the cell sample at 4 °C overnight. Antibodies were washed for 15 min three times, then Goat anti-Mouse Alexa Fluor™ Plus 488 antibody (A-11001) and Goat anti-Rabbit Alexa Fluor™ Plus 594 antibody (A-11012) were diluted at 1 : 300–1 : 500 ratio by blocking buffer. The cell sample was incubated in the secondary antibody buffer at room temperature for 1h–3h. Then the cell sample was washed for 15 min three times by 1 × TBST. The cell nuclear was stained by DAPI at 1 : 100 ratio for 30 min and washed. The data was obtained by NIKON A1 HD25 confocal microscope.
9
Immunofluorescence of Ovulated Oocyte
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The ovulated oocyte was fixed by 4% PFA in DPBS with 0.1% Triton-X 100 at 37 °C for 1h. Then oocytes were washed three times by blocking buffer containing 1 × TBST (leagene, PW0020) with 0.3% BSA (Sigma, A1933). Next, oocytes were incubated in a blocking buffer with 1 : 50 dilution of anti-α-tubulin (Cell Signaling Technology, 5063S) at 4 °C overnight. The anti-α-tubulin was washed three times by blocking buffer and stained with DAPI of 1 : 100 dilution. The image was obtained by NIKON A1 HD25 confocal microscope.
10
Tissue Clearing for Enhanced Imaging
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The PEGASOS method was used for tissue clearing, as described previously.(27 ) In short, the mice were deeply anesthetized and transcardially perfused with 0.1 M 1× PBS, followed by 4% PFA. Tissues were removed and postfixed overnight in 4% PFA. The skeletal tissues were decalcified with 0.5 M EDTA at 4°C for 3–5 days to improve the clearing effect. The EDTA solution was replaced daily with fresh solution. In the last phase of demineralization, the skeletal tissues were sectioned with a Leica cryostat to thin them to further improve the clearing effect. Then the decalcified bones and soft tissues were soaked in primary clearing buffer containing 40% tert‐butyl alcohol, 30% PBS, 20% Triton TMX‐100 (SLBZ8156, Sigma), and 10% ethanolamine (SHBG0568 V, Sigma) at 4°C for 3 days, and the buffer was replaced every day. The samples were immersed in secondary buffer with 50% tert‐butyl alcohol, 20% PBS, 20% Triton‐100, and 10% ethanolamine at 4°C for 2 days, which was replaced with fresh buffer daily. Finally, the samples were placed in the final clearing solution buffer of 70% tert‐butyl alcohol, 10% PBS, 20% Triton‐100, and 10% ethanolamine at 4°C for 1 day. These prepared tissues were then subjected to imaging with a Nikon A1 HD25 confocal microscope.
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