Cells were fixed with 4% PFA for 10 min, washed 3 times with PBS, and blocked with 10% goat serum (Victor, Cat#S-1000) in 0.2% PBST (0.2% Triton X-100 in PBS) for 1 hour at room temperature. After being washed 3 times with 0.2% PBST, cells were incubated with an anti-mouse Prox1 antibody (1:200) followed by Alexa flour 488 conjugated anti-rabbit antibody (1:500) at 4°C overnight. After 3 washes with 0.2% PBST, cells were mounted with Vector H-1000 (Vector, USA) and observed with fluorescent microscope (Olympus IX71).
Vector h 1000
Manufactured by Vector Laboratories
Sourced in United States
The Vector H-1000 is a high-performance spectrophotometer designed for accurate and efficient absorption measurements. It features a wide wavelength range, adjustable bandwidth, and advanced optics for reliable and consistent results.
Automatically generated - may contain errors
Lab products found in correlation
Cm1950, by Leica camera (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Anti mouse igg alexa fluor 488, by Cell Signaling Technology (1 mentions)
Alexa fluor 594 anti rabbit igg, by Cell Signaling Technology (1 mentions)
Lsm 700 confocal module, by Zeiss (1 mentions)
Foxp3, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Methylbutane, by Merck Group (1 mentions)
Agarose, by Merck Group (1 mentions)
Diaminophenylindole dapi, by Merck Group (1 mentions)
Fv1200mpe, by Olympus (1 mentions)
Sucrose, by Merck Group (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
A1r laser scanning confocal microscope, by Nikon (1 mentions)
Plan apo λ 100x oil objective, by Nikon (1 mentions)
Cm3050s cryomicrotome, by Leica camera (1 mentions)
Tcs sp8 confocal laser scanning microscope, by Leica camera (1 mentions)
Dihydroethidium, by Thermo Fisher Scientific (1 mentions)
Spinning disc confocal microscope, by Zeiss (1 mentions)
Axiovision software, by Zeiss (1 mentions)
14 protocols using vector h 1000
1
Prox1 Immunofluorescence Staining Protocol
2
Immunofluorescence Staining of FoxP3 and GrB
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Antigen retrieval was performed on deparaffinized sections in 10 mM citrate buffer (pH 6.0) in a pressure cooker for 15 min, followed by a wash in 0.1 M phosphate buffer (PB; pH 7.0). Subsequently, to quench any autofluorescence, the sections were treated with 50 mM glycine for 40 min at room temperature. Thereafter, non-specific staining was blocked with 1% bovine serum albumin for 30 min, followed by an overnight incubation at 4 °C with the primary antibodies against FoxP3 (Abcam, Cambridge, UK) and GrB (both antibodies at a 1:90 dilutions). Next, the sections were washed and incubated for 1 h with the secondary antibodies (1:300, Alexa Fluor 594 anti-rabbit IgG, Cell Signaling and 1:300 Alexa Fluor 488 anti-mouse IgG, Cell Signaling). Nuclei were counterstained with DAPI (1 μg/mL, Sigma-Aldrich, St. Louis, MO, USA). Finally, the sections were mounted in Vectashield (Vector H-1000, Vector). The samples were imaged with a Zeiss Axio Observer inverted microscope (×20 or ×40 NA 1.3 oil objectives) equipped with a Zeiss LSM 700 confocal module (Carl Zeiss Microimaging GmbH, Jena, Germany).
3
Cryopreservation and Imaging of Pancreatic Islets
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Pellets of the pancreatic islets were fixed using formaldehyde (4%, Sigma-Aldrich) overnight. The islets were then washed using PBS. Pellets were centrifuged (1 min at 1300 rpm), the supernatant was removed and agarose (2%, Sigma-Aldrich) was added. Pellets in the agarose were immediately centrifuged (1 min at 1800 rpm). After the agarose solidified, the pellets were transferred into sucrose (30%, Sigma-Aldrich) for overnight incubation at 4 °C. After incubation, the islets were transferred to Tissue-Tek (Sakura, Alphen aan den Rijn, Netherlands) and frozen in methylbutane (Sigma-Aldrich, St. Louis, MO, USA) cooled by liquid nitrogen. Frozen pellets were stored at −80 °C. Sections (20 μm) from the pancreatic islet pellets were cut using a cryomicrotome (Leica CM1950). The samples were stained with diamino-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA) and mounted with a vectashield (Vector H-1000, Burlingame, CA, USA) on a glass slide. For confirmation of the nanoparticle signal and its location, the Olympus FV1200MPE (Olympus life Science, Tokyo, Japan) confocal microscope was used (green background - Argon laser λ = 488 nm, DAPI - EPI lamp λ = 405 nm, ICG - LD599 laser λ = 647 nm). The images were taken using 20× (air) and 60× (oil immersion) objectives under 200× or 600× magnification respectively.
4
Progesterone Effect on Actin Polymerization
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To assess the effect of progesterone treatment on actin polymerization, SOECs were cultured in 3.5 cm Petri dishes (38.5 °C, 5% CO2 and humidified atmosphere) and treated with the three different P4 concentrations. Once they reached 50% and 100% of confluence, SOECs were fixed overnight in paraformaldehyde 4%, then washed three times with PBS for 5 min each prior to the incubation with TRITC-conjugated phalloidin 488 (50 μg/mL in PBS, dilution 1:100) for 1 h. After three washes with PBS for 5 min, cell samples were mounted with Vectashield mounting medium (Vector H-1000) and subjected to microscopy analysis.
5
Immunofluorescence Staining Protocol
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Cells were plated on 12 mm coverslips. After TGFβ treatment, cells were fixed for 10 min in 3.7% formaldehyde at room temperature for 20 min and permeabilised with 0.2% Triton X-100 for 5 min. Next, cells were washed 3 times with PBS and incubated for 5 min with 0.1 µg/ml DAPI. Finally, the coverslips were washed 3 times with PBS, twice with water and once with ethanol, before mounting them onto microscope slides with Vectashield (Vector H-1000). Cells were imaged on a DeltaVision Spectris wide field microscope fitted with a 37°C environment chamber (Solent Scientifi c, Segensworth, United Kingdom).
6
Sperm Triplicate Fluorescence Staining
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Sperm samples were fixed in absolute ethanol for at least 1 h at −20°C, spread on glass slides and air-dried. Next, they were incubated with TRITC-phalloidin 80 nM in PBS for 50 min followed by incubation with DAPI 0.2 mM in PBS for 15 min and then FITC-conjugated PSA at 50 μg/ml in PBS for 20 min. Finally, they were washed three times with tap water and mounted with Vectashield mounting medium (Vector H-1000).
The acquisition of triple staining was realized with Nikon A1r laser confocal scanning microscope, equipped with a Plan Apo λ 100X Oil objective, detector Galvano, with a pinhole size of 69 μM and a pixel size 0.04 um. We used an averaged 2 mode in channels series as follows:
The acquisition of triple staining was realized with Nikon A1r laser confocal scanning microscope, equipped with a Plan Apo λ 100X Oil objective, detector Galvano, with a pinhole size of 69 μM and a pixel size 0.04 um. We used an averaged 2 mode in channels series as follows:
Channel 1: FITC: λexc = 488 nm; λem = 525/50 nm, at 6.6% of the maximum laser power
Channel 2: DAPI: λexc = 404 nm; λem = 450/50 nm at 3% of the maximum laser power
Channel 3: TRITC: λexc = 561.5 nm; λem = 595/50 nm at 1.8% of the maximum laser power
7
Assessing Actin Polymerization in Sperm
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In order to assessing the effect of Aminopurvalanol A treatment on actin polymerization, the sperm samples were fixed in absolute ethanol for at least 1 h at −20°C and spread on microscope slides. After air-drying, they were incubated with FITC-conjugated phalloidin (3 μM in PBS) for 60 min, washed two times with distilled water and mounted with Vectashield mounting medium (Vector H-1000). For confocal microscopy set up refer to next paragraph.
8
Assessing Apoptosis in DEHP-Treated Testes
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DEHP-treated testis were fixed 4% paraformaldehyde for overnight at 4°C and embedded in Optimum Cutting Temperature (O.C.T.) compound (Sakura Finetek 4583). The embedded samples were sectioned using a CM3050S cryomicrotome (Leica) to a thickness of 10 μm. The sections were permeabilized, blocked, incubated with Anti-Cleaved Caspase-3 (CST 9661, RRID:AB_2341188 , 1:400 dilution) for overnight at 4°C, and then incubated with Anti-rabbit Alexa 488 (Invitrogen A21206, 1:500 dilution) and 1 μg/mL DAPI for 1 hr at room temperature. The sections were washed after primary and secondary antibody treatments. Samples were mounted using Vectashield (Vector H-1000) and observed under a TCS SP8 confocal laser scanning microscope (Leica).
9
Drosophila Brain Oxidative Stress Imaging
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Brains were dissected from adult flies at room temperature in Drosophila Schneider’s cell medium (SCM) supplemented with L-glutamine, then incubated in SCM with 30 μM dihydroethidium (Thermo Fisher Scientific, D11347) on a nutator for 7 min in the dark. Following a rinse with SCM, brains were washed 3 x 5 min with SCM, then placed on a glass slide between double-sided tape strips and covered with coverglass. Vectashield mounting medium (Vector #H-1000) was infused under the coverglass and brains were immediately scanned at 10x magnification using a Zeiss LSM 780 confocal laser-scanning microscope. Maximum intensity projections were created with ImageJ software.
10
Immunofluorescence Staining and Imaging Protocol
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