Tcs sp2 aobs confocal laser scanning microscope

Cells were plated in fibronectin (3 μg/mL)-coated 24-well plates, and fixed 10 min with ice-cold 4% of PBS paraformaldehyde (Santa Cruz Biotechnology, Dallas, TX, USA). A total of 0.1% of Triton X-100 and 1% of BSA were used for permeabilization and saturation, respectively. Then the cells were incubated with a primary antibody (Table S2) and Alexa Fluor 488-conjugated anti-goat/rabbit secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. TCS SP2 AOBS confocal laser-scanning microscope and LAS AF software (Leica Microsystems, Wetzlar, Germany) were used to obtain immunofluorescence images. Immunofluorescence quantification was performed using the ImageJ software. Green (number of replicates = 6) and yellow (number of replicates = 3/4) positive cells were counted and normalized on nuclei number (DAPI staining).

Hek293T cells were plated in fibronectin (6 µg/mL)-coated 24-well plates and fixed for 10 min with ice-cold 4% PBS paraformaldehyde (Santa Cruz Biotechnology, Dallas, TX, USA). A total of 0.1% of Triton X-100 and 1% of BSA was used for permeabilization and saturation, respectively. Then, the cells were incubated with a primary antibody (Table S2) for 1 h at room temperature. Incubation with secondary antibodies was performed using a specific Alexa 488 (green) or 555 (red)-conjugated anti-goat/rabbit/mouse secondary antibody (Invitrogen, Waltham, MA, USA) for 1 h at room temperature. Duolink in situ mounting medium (Sigma Aldrich) with DAPI was used for nuclear staining and mounting. A TCS SP2 AOBS confocal laser-scanning microscope and the LAS AF v2.6 software (Leica Microsystems, Wetzlar, Germany) were used to obtain immunofluorescence images.

For VDR detection, purified Vγ9Vδ2⁺ T cells were treated with 1α,25(OH)₂D₃ (50 nM) for 12 hours and washed once by PBS. In some experiments, vehicle or 1α,25(OH)₂D₃ (50 nM) pretreated Vγ9Vδ2 T cells were stimulated with anti-human CD3 (5 µg/mL) and anti-human CD28 (1 µg/mL), or PMA (50 ng/mL) plus ionomycin (1 µg/mL) at the indicated time points (0 s, 120 s, 60 min, and 4 hours). Subsequently, T cells were fixed in 4% (vol/vol) paraformaldehyde for 30 min in room temperature, cells were permeabilized with 0.2% Triton X−100 (Sigma) for 30 min, and blocked in Tris Buffered Saline (TBS) (0.02% Triton X-100% and 2% BSA in PBS) for another 2 hours. Then T cells were incubated with anti-VDR antibody (Santa Cruz Biotechnology, sc13133) at 4°C overnight. After three washes with PBS, the cells were incubated at room temperature with Alexa Fluor 488 goat anti-mouse IgG (H+L) (Thermo Fisher, CA11005S) secondary antibody (1:200 dilution) for another 2 hours. Negative controls were carried out using isotype IgG. Subsequently, the cells were counterstained with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) for 5 min in the dark. All images were captured with a Leica TCS SP2 AOBS confocal laser scanning microscope (Leica Microsystems).

Sections of all above-described animals were fixed and stained as described above, excluding the endogenous peroxidase blocking step, ABC step and DAB visualisation. Primary antibodies are listed in Table 1. Secondary antibodies used were donkey anti-goat and donkey anti-rabbit, both coupled to either Alexa 488 or Alexa 594, (dilution 1:400, Invitrogen, Camarillo, CA, USA). For the detection of β-pleated sheets, sections were incubated with 1% Thioflavin S (ThioS, Sigma, St. Louis, Missouri USA) in milliQ for 5 minutes, washed three times with 70% ethanol and two times with TBS. Sections were mounted with Vectashield® (Vector laboratories Inc) or PVA-DABCO^® mounting medium (Sigma). Between incubation steps, sections were washed extensively with TBS. A Leica TCS SP2 AOBS confocal laser scanning microscope (Leica Microsystems) was used to visualise the immunofluorescence. To exclude bleed-through of fluorescence emission, a series of images was obtained by sequential scanning of channels through a 40× lens (zoom factor 1× or 2×, resolution 1024 × 1024). Staining was performed and analyzed on at least three sections per animal. In the figures, a representative image of these stainings is shown.

Immunostainings were visualized using Leica TCS-SP2 AOBS confocal laser scanning microscope equipped with 63×/1.40 HCX PL APO oil-immersion objective (Leica Microsystems, Wetzlar, Germany). Confocal images shown are calculated maximum projections of sequences of three proximate single sections. SIM analysis was carried out on singularized spermatids from testis suspensions, which were fixed with 2% formaldehyde in PBS and spread on slides. After gentle drying, samples were further processed for immunolabeling as described above. SIM was conducted using Zeiss ELYRA S.1 equipped with Zeiss Plan-Apochromat 63×/1.4 oil DIC objective and run with Zeiss ZEN 2012 software package including Superresolution Structured Illumination plugin (Carl Zeiss Microscopy GmbH, Jena, Germany). Images were processed with ImageJ (National Institutes of Health, version 1.42q; http://rsbweb.nih.gov/ij) and Adobe Photoshop CS5 (Adobe Systems).

TOV112D cells were plated in 8 wells Nunc™ Lab-Tek II™ Chamber Slides™. Immunofluorescence experiments were carried out as previously described [35 (link)]. Images were acquired by the Leica TCS SP2 AOBS confocal laser-scanning microscope (Leica Microsystems).

Confocal microscopy was performed with a Leica TCS SP2 AOBS confocal laser-scanning microscope (Leica Microsystems, Buffalo Grove, IL) with HC PL APO 20× (NA 0.7), HCX PL APO 60× (NA 1.2), and HCX PL APO 100× (NA 1.4) objective lenses (Leica Microsystems). CFW and aniline blue fluorescence was recorded by excitation with a 405-nm blue diode laser and detection of emission fluorescence between wavelengths of 415 and 520 nm. GFP, cDFFDA, and Alexa Fluor 488 fluorescence was recorded by excitation with a 488-nm Ar/Kr laser and detection of 500–550 nm emission fluorescence. TagRFP and MitoTracker Red fluorescence was recorded by excitation with a 543-nm He/Ne laser and detection of 580–650 nm (TagRFP) or 555–650 nm (MitoTracker Red) emission fluorescence. Images were captured by Leica Confocal software (Leica Microsystems). Images with different fluorescence channels were overlaid using Image J software (rsbweb.nih.gov/ij/), or DP Manager software (Olympus, Tokyo, Japan) when relative positions of images needed manual adjustment.