FISH was performed as previously described in Ref. [21] (link). Slides were analyzed by epifluorescence microscopy (Leica, Nanterre, France).
Epifluorescence microscopy
Manufactured by Leica
Sourced in Germany
Epifluorescence microscopy is a technique that uses fluorescence to visualize microscopic specimens. The core function of this equipment is to illuminate the sample with a specific wavelength of light, causing fluorescent molecules within the sample to emit light at a different wavelength, which is then detected and captured by the microscope's optics and imaging system.
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Lab products found in correlation
Triton x 100, by Merck Group (2 mentions)
Paraformaldehyde (pfa), by BioLegend (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Hoechst 33420, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti cip2a antibody, by Santa Cruz Biotechnology (1 mentions)
Dylight 488 conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (1 mentions)
Lsm 880, by Zeiss (1 mentions)
Axio scan z1, by Zeiss (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Phalloidin tritc, by Merck Group (1 mentions)
Facs fortessa, by BD (1 mentions)
Ph rodamine, by Thermo Fisher Scientific (1 mentions)
10 protocols using epifluorescence microscopy
1
Fluorescence In Situ Hybridization (FISH)
2
Cell Orientation Analysis by Fluorescence Microscopy
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Cells were fixed with 4% paraformaldehyde (Biolegend, San Diego, USA) for 10 min at room temperature, followed by permeabilization using 0.1% Triton X100 (Merck KGaA, Darmstadt, Germany) for 10 min at room temperature. Afterward, cells were stained 24 h with Hoechst‐33420 for the cell nucleus (dilution 1:10 000 in PBS; Thermo Fisher Scientific, Dreieich, Germany) and phalloidin conjugated with Alexa Fluor 488 for the actin cytoskeleton (dilution 1:250 in PBS; Thermo Fisher Scientific, Dreieich, Germany). Images were gathered by epifluorescence microscopy (Leica, Wetzlar, Germany) using a 20× LD objective (NA 1.3; Leica, Wetzlar, Germany). Cell orientation was analyzed using the OrientationJ plug‐in[64 ] of ImageJ (NIH, Bethesda, USA), as validated by Xu et al.[66 ] The cell orientation index was in the range between 0 and 1, where 0 corresponds to randomly oriented cells, and 1 corresponds to a perfectly oriented cell. The quantification was performed at four randomly selected positions of each sample from four independent samples.
3
Evaluating Sperm-ZP Binding and Acrosome Integrity
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BZP (50–55) conjugated with ZP2C, ZP3, ZP4 were washed twice in TALP medium and co-incubated (38.5 °C, 5% CO2, 20% O2 and saturated humidity) with double centrifuged spermatozoa (200,000 sperm/mL) in 500 μl medium. In addition, BZP2C were inseminated with sperm prepared by swim-up (200,000 sperm/mL) to validate BZP2C as a suitable model for sperm-ZP recognition independent of the method of sperm preparation. To assess the number of sperm bound and the integrity of their acrosomes after 0.5, 1, 2, and 20 h of coincubation, an aliquot of BZP2C was washed three times in PBS, supplemented with 0.1% PVA, fixed with 0.5% glutaraldehyde in PBS (v/v), and stained with 0.01 mM Hoechst 33342, 4 μg/mL fluorescein isothiocyanate-conjugated peanut agglutinin (PNA-FITC) and 20 μg/mL of propidium iodide (PI). BZP were mounted on slides, and the number of sperms bound and acrosome status (acrosome from reacted sperm stains green), were recorded under an epifluorescence microscopy (Leica, DMLS, Barcelona, Spain). The percent of beads with at least one sperm bound (BZPSB) and the mean number of sperm per bead (S/BZP) were recorded and acrosome integrity was evaluated. Spermatozoa with damaged acrosome and acrosomal content were observed green. Three replicates were performed.
4
Immunofluorescence Analysis of AXL and DAPK Signaling
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Cells treated with or without GAS6 were plated on a glass slide and fixed in 4% paraformaldehyde and 0.2% Tween 20 in PBS. After a brief wash in PBS, the slides were blocked with 5% normal serum in 1% BSA/0.2% Triton X-100 for 1 h and incubated with mouse monoclonal anti-CIP2A antibody (1:50; Santa Cruz), rabbit polyclonal anti-p-AXL antibody (1:40; R & D) and mouse polyclonal anti-p-DAPK antibody (1:50; Biorbyt). After an overnight incubation, the slides were washed and then incubated with fluorescent (Alexa 568, Alexa 488) conjugated secondary antibodies (1:200) for 1 h and counterstained for nuclei with ToTo-3. The stained slides were mounted and analysed by epifluorescence microscopy (Leica). Pictures were captured using a Photometrics CoolSNAP EZ system (high-performance EMCCD and CCD Cameras) and MetaMorph version software (Molecular Devices).
5
Immunostaining of Olig2-Expressing Cells
6
Whole Brain Fluorescence Imaging
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Fluorescence-labeled whole brains sections (RNAscope or tdTomato) were scanned by the Whole Brain Microscopy Facility at UTSouthwestern (see Acknowledgments) using a Zeiss Axioscan. Z1 and appropriate filters. We also used a confocal microscope (Zeiss LSM880) available at the Quantitative Light Microscopy Core (see acknowledgments) to image fluorescence-labeled tissues at higher magnifications. ImageJ (NIH, Bethesda, MD, USA) and Adobe Photoshop 2021 were used to uniformly adjust the resolution and contrast of all our digital images. Estimates of signal strengths were done by visually inspecting brain sections under epifluorescence microscopy (Leica, Teaneck, NJ, USA). Brain sites were visually identified and ranked by expression level with reference to the Franklin and Paxinos atlas (3rd edition).
7
Fluorescent Imaging of Cells
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epifluorescence microscopy allowed the observation of nucleus and actin cytoskeleton of adherent cells respectively by staining with 4′,6′-diamidino-2-phenylindole (DAPI) molecule and Alexa Fluor™ 488 phalloidin (Thermo Fisher Scientific Inc., Essone, France).
Cells were fixed with paraformaldehyde solution (4%) for 15 min and then rinsed during 5 min with PBS to remove non-adherent cells, and incubated for 15 min in a PBS solution containing 0.5% Triton™ X-100 to permeabilize them. This reaction is stopped by two successive washes with PBS.
The marking was carried out by diluting 50 μl of phalloidin, 50 μl of Triton ™ X-100 and 0.025 g of bovine serum albumin (BSA) in 5 ml of PBS, this mixture is added to cells and is left to act 30 min. After incubation, three successive washes with PBS were performed.
For nuclei marking, DAPI solution was used by diluting of 5 μl of Triton™ X-100 and 5 μl of DAPI in 5 ml of PBS. Contact time was 20 s followed by 2 PBS rinses. Cells were covered with 100 μl of PBS before visualization by epifluorescence microscopy (Leica, Germany). The 12well plates were protected from light throughout the preparation.
Cells were fixed with paraformaldehyde solution (4%) for 15 min and then rinsed during 5 min with PBS to remove non-adherent cells, and incubated for 15 min in a PBS solution containing 0.5% Triton™ X-100 to permeabilize them. This reaction is stopped by two successive washes with PBS.
The marking was carried out by diluting 50 μl of phalloidin, 50 μl of Triton ™ X-100 and 0.025 g of bovine serum albumin (BSA) in 5 ml of PBS, this mixture is added to cells and is left to act 30 min. After incubation, three successive washes with PBS were performed.
For nuclei marking, DAPI solution was used by diluting of 5 μl of Triton™ X-100 and 5 μl of DAPI in 5 ml of PBS. Contact time was 20 s followed by 2 PBS rinses. Cells were covered with 100 μl of PBS before visualization by epifluorescence microscopy (Leica, Germany). The 12well plates were protected from light throughout the preparation.
8
Immunofluorescence Staining of α-SMA and F-Actin
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Cells grown in 4-well CultureSlides were fixed with cold 4% paraformaldehyde for 15 min and permeabilized with 0.5% Triton X-100 for 30 min. Once blocked with 1% bovine serum albumin-PBS for 1 h, a primary antibody against α-SMA (Sigma, 1:500) was added for 1 h at 37 °C. A suitable fluorescent secondary antibody and Phalloidin-TRITC (Sigma) to detect F-actin were also incubated for 1 h. Cells were counterstained with DAPI (1:10.000) and mounted with ProlongTM Gold antifade reagent (Thermo Fisher Scientific, Waltham, MA, USA). Epifluorescence microscopy (Leica Microsystems, Wetzlar, Germany) was used to image the immunostainings with a 20× objective. Processing and analysis of immunofluorescence images were carried out with ImageJ/Fiji software [57 (link)]. F-actin and α-SMA images were background subtracted and used to compute the total intensity, which was normalized by the corresponding total cell number and averaged for each patient and condition.
9
Quantifying Myelin Phagocytosis by Myeloid Cells
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To assess the capacity of myeloid cells to ingest myelin, we first incubated the purified human myelin with a pH-sensitive dye (pH-Rodamine; Invitrogen) for 1 h in PBS (pH = 8). Fluorecently-labeled myelin was then added at a final concentration of 20 μg/mL to microglia or macrophage that were pre-exposed to distinct B-cell supernatants for two days. Following 1 h of incubation at 37 °C, epifluorescence microscopy (Leica Microsystems, Wetzlar, Germany) was used to visualize and quantify myelin phagocytosis by the microglia and macrophages, and flow cytometry (FACS Fortessa, BD Bioscience) was used to quantify internalized myelin. Macrophage myelin phagocytosis assays were performed to directly compare the impact of pro-inflammatory versus IL-10 expressing B cell supernatants derived from both RRMS patients and healthy controls (HC) on responses of the same HC donor macrophage.
10
Comet Assay for DNA Damage
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Assays were performed according to the manufacturer's introduction (Jiancheng, Nanjing, China). Briefly, the indicated cells were mixed with agarose on the assay slides and after solidification, the slides were immersed in the lysis solution for 2 hours at 4 ℃. Thereafter, the slides were placed in unwinding solution for 1 hour at room temperature before submersion in the lysing solution for 2 h. The slides were then electrophoresed in electrophoresis buffer at 25 V (adjusted to 300 mA) for 30 min, neutralized with Tris buffer before staining with PI and observation by epifluorescence microscopy (Leica Microsystems, Germany).
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