The presence of OASCF was assessed by detection of PLCζ. Sperm specimens were smeared on glass slides, fixed with recently prepared 4% paraformaldehyde solution/PBS for 20 min, and washed with 0.1% Tween 20/PBS for 5 min. Slides were permeabilized with 1% Triton X-100/PBS for 1 h at room temperature. To block unspecific binding sites, 5% of normal goat serum/PBS was added on the specimen for 1 h at 37 °C. The specimens were then incubated overnight with anti-rabbit PLCζ antibody (pab0367, Covalab, Bron, France) in 5% normal goat serum/PBS at 4 °C. After rinsing in PBS, slides with specimens were incubated with FITC-coated goat anti-rabbit IgG (ab150077, Abcam, Cambridge, UK), which cross-reacts with human PLCζ. Lastly, spermatozoa were counterstained with 10 μg/ml 4′,6-diamino-2-phenylindole (DAPI/Antifade Solution, Millipore, Temecula, CA, USA) and visualized under a fluorescent microscope to assess the percentage of spermatozoa exhibiting PLCζ fluorescence in the acrosomal, equatorial, and post-acrosomal regions of the sperm head. At least 200 spermatozoa were counted for each specimen. By assessing a large number of patients for PLCζ, we found that those with less than 30% PLCζ yielded consistently low or unobtainable fertilization. Therefore, the threshold of 30% was adopted [20 (link)].
Dapi antifade solution
Manufactured by Merck Group
Sourced in United States, Germany
DAPI/Antifade solution is a laboratory reagent used in fluorescence microscopy. It contains the fluorescent dye DAPI, which binds to DNA, and an antifade agent to prevent photobleaching. The solution is used to stain and preserve DNA samples for imaging and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780, by Zeiss (2 mentions)
Rubber cement, by MP Biomedicals (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Ab150077, by Abcam (1 mentions)
Chloroquine, by Bio-Techne (1 mentions)
Fitc conjugated goat anti rabbit igg, by Merck Group (1 mentions)
Vybrant fam caspase 3 and 7 assay kit, by Thermo Fisher Scientific (1 mentions)
3 4 5 dimethyl 2 thiazolyl 2 5 diphenyl 2h tetrazolium bromide mtt, by Merck Group (1 mentions)
Celltracker green cmfda, by Thermo Fisher Scientific (1 mentions)
Discovery bio wide pore c5, by Merck Group (1 mentions)
Hplc station, by Bio-Rad (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Matrigel, by BD (1 mentions)
Pdsred2 er vector, by Takara Bio (1 mentions)
Anti laminin, by Abcam (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Anti fibronectin, by Merck Group (1 mentions)
Zoe fluorescent cell imager, by Bio-Rad (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Mouse anti α sma, by Abcam (1 mentions)
9 protocols using dapi antifade solution
1
Sperm Acrosomal PLCζ Evaluation
2
Lactaptin RL2 Characterization and Purification
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The following reagents were used: Matrigel (BD Bioscience), DAPI/Antifade solution (CHEMICON International, Inc), Cell Tracker Green CMFDA (Invitrogen, Tokyo, Japan), Vybrant FAM caspase-3 and -7 assay kit (Molecular Probes), MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (Sigma-Aldrich), DAPI/Antifade solution (Millipore, Temecula, CA), chloroquine (Tocris Bioscience), complete protease inhibitor cocktail (Roshe Diagnostics,Germany), PMSF (Sigma-Aldrich). Molecular weight markers were from Thermo Scientific: 14.4–116.0 kDa (#26610) and 10–250 kDa (#26619).
Antibodies against human LC3A/B, MDM2, Bax and tubulin were from Abcam, p53 and Bcl-2 were from Sigma-Aldrich, and anti-RL2 was from BioSan-R (Novosibirsk, Russia). The secondary antibodies used in this study were HRP-conjugated goat anti-mouse and goat anti-rabbit (BioSan-R, Novosibirsk, Russia) or FITC-conjugated goat anti-rabbit IgG (Sigma-Aldrich).
The recombinant analogue of lactaptin RL2 was obtained from E.coli and purified as described previously [3] (link). The 98% purity of the isolated protein was confirmed by RP-HPLC chromatography on C5 reverse phase column (Discovery BIO Wide Pore C5, Sigma) in water (0.5% TFA)-acetonitil solvent system using HPLC Station (Bio-RAD Laboratories) as well as by RP-HPLC on C18 (ProntosSIL) using Milichrom A-02 station (EcoNova, Russia).
Antibodies against human LC3A/B, MDM2, Bax and tubulin were from Abcam, p53 and Bcl-2 were from Sigma-Aldrich, and anti-RL2 was from BioSan-R (Novosibirsk, Russia). The secondary antibodies used in this study were HRP-conjugated goat anti-mouse and goat anti-rabbit (BioSan-R, Novosibirsk, Russia) or FITC-conjugated goat anti-rabbit IgG (Sigma-Aldrich).
The recombinant analogue of lactaptin RL2 was obtained from E.coli and purified as described previously [3] (link). The 98% purity of the isolated protein was confirmed by RP-HPLC chromatography on C5 reverse phase column (Discovery BIO Wide Pore C5, Sigma) in water (0.5% TFA)-acetonitil solvent system using HPLC Station (Bio-RAD Laboratories) as well as by RP-HPLC on C18 (ProntosSIL) using Milichrom A-02 station (EcoNova, Russia).
3
Subcellular Localization of FX Protein
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To determine the subcellular localization of the WT or mutant FX protein, a fluorescent antibody and two red fluorescent protein vectors that were designed to specifically label the endoplasmic reticulum and Golgi apparatus, respectively, were used. HEK293 cells grown on cover slips were transiently co-transfected with 1 μg of each plasmid constructs [pcDNA3.1(-), pcDNA/FX-WT, or pcDNA/FX-A275V] and 1 μg of pDsRed-Monomer-Golgi vector or pDsRed2-ER vector (Clontech Laboratories, Mountain View, CA, USA) using a PolyFect reagent. The cells were fixed in 4% paraformaldehyde for 15 min at room temperature, washed three times in PBS, permeabilized in 1% Triton X-100/ PBS, and blocked using 1% BSA in PBS. Monoclonal mouse antihuman FX antibody were incubated at 4 °C overnight, and then fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (H + L) (KPL,Gaithersburg, MD, USA) was added, followed by incubation for 1 h at room temperature. Then, the cover slips were mounted onto glass slides with DAPI/anti-fade solution (Millipore, MA, USA), followed by capturing fluorescence images using a confocal laser scanning microscope (Zeiss, LSM780, Germany).
4
Extracellular Matrix Protein Characterization
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dECM samples were prepared on coverslips as described above and then fixed with 3.7% formalin (Sigma-Aldrich, St. Louis, MO, USA) for 15 min, permeabilized with 0.5% Triton-X100 (Sigma-Aldrich, St. Louis, MO, USA) for 15 min, blocked with 1% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) and stained overnight at +4 °C with the following primary antibodies: anti-fibronectin (number F3648, Sigma-Aldrich, St. Louis, MO, USA), anti-collagen type III (number RAH C33) and anti-collagen type IV (number RAH C44, both from Imtek, Moscow, Russia), and anti-laminin (number ab11575, Abcam, Cambridge, UK). Next, the coverslips were washed with PBS three times for 15 min, stained with secondary Alexa Fluor™ 488-conjugated goat anti-rabbit antibodies (number A-11008, Thermo Fisher Scientific, Waltham, MA, USA) and mounted with DAPI/Antifade Solution (Sigma-Aldrich, St. Louis, MO, USA). Antibodies were diluted according to the recommendations of the manufacturers. Observation of the stained samples and image acquisition were performed with the use of the ZOE Fluorescent Cell Imager (Bio-Rad Laboratories Inc., Hercules, CA, USA).
5
Immunofluorescent Staining of Collagen-1 and αSMA
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BMDMs were fixed and permeabilized with pre-cold methanol on ice for 15 min. The cells were incubated with rabbit anti-collagen-1 (1:200, Abcam) and mouse anti-αSMA (1:400, Cat: ab7817, Abcam) at 4 °C overnight, followed by incubation with Alexa Fluor 488 goat anti-ribbit IgG (1:500, Invitrogen) and Alexa Fluor 594 goat anti-mouse IgG (1:500, Cat: A11005, Invitrogen) for 1 h.
Blood and BM cells from young and aged mice with subretinal fibrosis were fixed and permeabilized with pre-cold methanol on ice for 10 min. The samples were blocked and incubated with rat anti-mouse CD45 and rabbit anti-mouse collagen-1 followed by Alexa Fluor 594-conjugated goat anti-rabbit IgG and Alexa Fluor 488-conjugated donkey anti-rat IgG as described above. Cells were then re-suspended in 350 µl stain buffer and transferred onto slides using cytospin ROTOFIX 32A (Hettich, Kirchingen, Germany).
All samples were mounted with 4′,6-Diamidino-2-phenylindole (DAPI)/Anti-fade solution (Cat: S7113, Sigma Aldrich) and imaged using the Zeiss LSM 880 Confocal Microscope (Zeiss, Braunschweig, Germany).
Blood and BM cells from young and aged mice with subretinal fibrosis were fixed and permeabilized with pre-cold methanol on ice for 10 min. The samples were blocked and incubated with rat anti-mouse CD45 and rabbit anti-mouse collagen-1 followed by Alexa Fluor 594-conjugated goat anti-rabbit IgG and Alexa Fluor 488-conjugated donkey anti-rat IgG as described above. Cells were then re-suspended in 350 µl stain buffer and transferred onto slides using cytospin ROTOFIX 32A (Hettich, Kirchingen, Germany).
All samples were mounted with 4′,6-Diamidino-2-phenylindole (DAPI)/Anti-fade solution (Cat: S7113, Sigma Aldrich) and imaged using the Zeiss LSM 880 Confocal Microscope (Zeiss, Braunschweig, Germany).
6
LC3B-h Expression in LPS-Treated Cells
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The sequence of LC3B-h was inserted into modified pmCherry vector. Cells were cultured on coverslips with LPS (1.0 μg/ml) treatment. Cells were removed from the medium and rinsed with PBS. After incubating with 0.5% TritonX-100 at room temperature for 15 min, cells were fixed in 4% paraformaldehyde. The cells were washed in PBS, treated with 100% ethanol, and then air-dried. Cell nuclei were stained by 50 µl DAPI/Antifade solution (Sigma-Aldrich). Rubber cement (MP Biomedicals) was used for sealing coverslips, which were observed under the laser scanning confocal microscope afterward.
7
Immunofluorescence and Erythrocyte Imaging
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RAW macrophage cells were cultured on coverslips and after 48 h post-treatment washed with PBS. Subsequently, cells were incubated in PBS solution containing 4% paraformaldehyde for fixation at RT for 15 min. Following washing with cold PBS fixed cells were permeabilized by addition of PBS solution containing 0.2% Triton X-100 for 10 min at RT and subsequently blocked with 1% BSA in PBS solution. After blocking, respective primary antibodies (PBS with 1% BSA) were added to cells at RT for 1 h, followed by incubation with respective secondary antibodies. The stained cells after washing were mounted using DAPI antifade solution (Sigma-Aldrich) and analysed through a confocal microscope (Olympus Corporation, Japan). Image analysis was performed using NIS Elements software.
For the observation of eryptotic erythrocytes, loaded erythrocytes were washed and stained with FITC-Annexin-V (Thermo Scientific) at 1:20 dilution in Annexin-V binding Buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, and pH 7.4) for 20 min. After washing, mounted erythrocytes were analysed using confocal microscope (Nikon, Japan). 10 kDa FITC-dextran loaded erythrocytes were evaluated for loading efficiency by fluorescence microscope.
For the observation of eryptotic erythrocytes, loaded erythrocytes were washed and stained with FITC-Annexin-V (Thermo Scientific) at 1:20 dilution in Annexin-V binding Buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, and pH 7.4) for 20 min. After washing, mounted erythrocytes were analysed using confocal microscope (Nikon, Japan). 10 kDa FITC-dextran loaded erythrocytes were evaluated for loading efficiency by fluorescence microscope.
8
Fluorescent in situ Hybridization for circHADHA and miR-361
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Cells were cultured on coverslips with 1.0 μg/ml of lipopolysaccharide (LPS) treatment. Cells were removed from the medium and rinsed with phosphate buffered saline (PBS). After incubating with 0.5% TritonX-100 at room temperature for 15 min, cells were fixed in 4% paraformaldehyde. The cells were washed in phosphate buffered saline (PBS), treated with 100% ethanol, and then air-dried. Briefly, digoxin-labeled probes against circHADHA (5’, 3’ fluorescein isothiocyanate (FITC)-labeled) and miR-361 (5’, 3’ Cy3-labeled) targets (Geneseed Biotech) were denatured at 85°C for 5 min and hybridized at 37°C overnight. On the following day, the slides were washed in 2× saline-sodium citrate buffer (SSC, Sigma-Aldrich). Subsequently, blocking was performed with 3% bovine serum albumin (BSA) at 37°C for 30 min, and the anti-digoxigenin fluorescence-conjugated antibodies were added to the slides at 37°C for 1 h. After washing in PBS, the cell nuclei were stained by 50 µl DAPI/Antifade solution (Sigma-Aldrich). Rubber cement (MP Biomedicals) was used for sealing coverslips, which were observed under the laser scanning confocal microscope afterward.
9
Immunofluorescence Microscopy of γH2AX
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Cells attached to coverslips were washed once with cold PBS and fi xed with ice cold methanol-acetone (ratio 1:1) for 15 min, then blocked with 5% bovine serum albumin (fraction V, Sigma-Aldrich Chemie GmbH) in PBS for 60 min at room temperature then incubated with 1:500 dilution of the primary Alexa Fluor® 488 anti-H2AX-Phosphorylated (Ser139) Antibody (BioLegend), at 4 ºC overnight. After fi ve washings in PBST, lasting 3 min each, cells were counterstained with Dapi-antifade solution (Sigma-Aldrich Chemie GmbH). The visualization of cells was done using two microscopes: AxioVision (Carl Zeiss) comprising ApoTome software and AxioImager A1 (Carl Zeiss) with a 100 x objective. All images were captured using AxioVision software. Before the observation of foci, digital images were processed with ImageJ to adjust brightness and contrast.
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