Alexa fluor 594 igg

Immunofluorescence assays of log-phase promastigotes, heat-shocked promastigotes and axenic amastigotes were performed as described previously [38 (link)]. Briefly, 2 × 10⁵ cells were washed with 1×PBS and applied on microscopic slides and fixed with ice-cold methanol. Following permeabilization and blocking, the cells were stained with primary anti-HA IgG antibody (polyclonal, mouse, 1:3000; Invitrogen, Carlsbad, CA, USA) and secondary anti-mouse Alexa Fluor^® 594 IgG (polyclonal, goat, 1:1000; Thermo Fisher Scientific, Waltham, MA, USA) and DAPI (1:50; Sigma Aldrich, Munich, Germany). Fluorescence microscopy was carried out on an EVOS^® FL Auto Cell Imaging System using a 64× magnification.

Cells were seeded at a density of 3.5 × 10³ cells/spot on spot slides and grown in 50 µL of complete cellular medium. The next day, cells were infected with wild-type H-1PV at an MOI of 500 pfu/cell in a total of 70 µL of 5% fetal bovine serum (FCS)-containing medium. At 2 h post-infection, cells were fixed with 3.7% paraformaldehyde on ice for 15 min and permeabilised with 1% Triton X-100 for 10 min. Immunostaining was carried out with the following antibodies, all used at 1:500 dilution for 1 h: mouse monoclonal anti-H-1PV capsid [29 (link)] and rabbit polyclonal anti-galectin-1 (HPA000646; Sigma-Aldrich, Darmstadt, Germany). Anti-mouse Alexa Fluor 594 IgG (A11005; Thermo Fisher Scientific, Carlsbad, CA, USA) or anti-rabbit Alexa Fluor 488 IgG (A11008; Thermo Fisher Scientific, Carlsbad, CA, USA) were used as secondary antibodies. Nuclei were stained with DAPI. Images of randomly assigned cells in the green channel (galectin-1), red channel (H-1PV), or blue channel (DAPI) were acquired with a confocal microscope (Leica TCS SP5 II, Wetzlar, Germany). Picture analysis was carried out using the LAS X Software (Leica, Wetzlar, Germany).

Human and murine lung tissue sections were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) and embedded in paraffin. Sections were deparaffinized and hydrated followed by antigen retrieval by boiling in 0.01 M citrate buffer (pH 6.0) for 10 min. Lung sections were blocked with 3% normal donkey serum (Sigma-Aldrich, St. Louis, MO) in PBS for 20 min followed by incubation with antibodies: anti-SP-A (Santa Cruz), PARP (Cell Signaling), p45 NFE2 or DJ-1 overnight. The secondary antibodies, Alexa Fluor 594 IgG, Alexa Fluor 488 IgG and Alexa Fluor 647 IgG (Thermo Fisher Scientific, Waltham, MA) were applied for 1 h. Sections were mounted with Fluoroshield Mounting Medium containing DAPI (Abcam, Cambridge, MA) and analyzed using Zeiss Axio Imager Confocal Fluorescent Microscopy.

Frozen kidney tissues and cells were transferred to 4% paraformaldehyde and fixed at 4 °C overnight. Cells and 4-μm tissue sections were permeabilized with 0.3% Triton X-100 for 10 min and blocked with 5% donkey serum for 1 h. They were then incubated with the following primary antibodies: anti-MAD2B (1:200, ab180579, Abcam, Cambridge, MA, USA), Numb (1:50, sc-136554, Santa Cruz, CA, USA), anti-Synaptopodin (1:200, #163004, Synaptic Systems, Gottingen, Germany), and anti-Nephrin (1:100, AF3159, R&D, Minneapolis, MN, USA) overnight at 4 °C. Alexa Fluor 488 IgG and Alexa Fluor 594 IgG (1:200, Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies. Nucleus was counterstained with Hoechst (Beyotime Biotechnology, Shanghai, China). Sections were observed under fluorescence microscope and images were captured at identical microscopic settings.

The sections were double stained with BrdU and NeuN or BrdU and GFAP and then incubated for 2 h in a mixture of mouse monoclonal anti-BrdU (1:150, BMC9313, Roche Molecular Biochemicals, Mannheim, Germany) and either rabbit polyclonal anti-NeuN (Neuronal nuclei; 1:500, ABN78, Millipore, Bedford, MA, USA) or goat polyclonal anti-GFAP (Glial fibrillary acidic protein; AB7260 1:200, Abcam Cambridge, MA, USA). This was followed by a 2-h incubation in a mixture of goat anti-mouse Alexa Fluor® 594 IgG (1:500, A11005, Invitrogen, Carlsbad, CA, USA) (BrdU) and goat anti-rabbit Alexa Fluor® 488(1:500, A11008, Invitrogen, Carlsbad, CA, USA) (NeuN, GFAP) at room temperature. Fluorescence signals were confirmed using a Zeiss LSM 710 confocal imaging system (Carl Zeiss, Oberkochen, Germany) with a sequential scanning mode for Alexa 594 and 488. Stacks of images (1024 × 1024 pixels) from consecutive 0.9–1.2-μm-thick slices were obtained by averaging eight scans per slice. The resulting images were processed with ZEN 2010 (Carl Zeiss).

Human lung tissue obtained from non-smokers (NS), smokers (SM), and emphysema patients (EM) was fixed in 4% paraformaledyde and embedded in paraffin. Sections were deparaffinized and hydrated followed by antigen retrieval by boiling in 0.01 M citrate buffer for 10 min and blocking with 3% normal donkey serum (Sigma-Aldrich, St. Louis, MO) for 20 min. Lung tissue sections were incubated with surfactant protein C (SP-C; Santa Cruz Biotechnology, CA), proSP-C (Millipore, Billerica MA), γH2AX, caspase 3 or 53BP1 overnight followed by adding corresponding fluorochrome-labeled secondary antibodies: Alexa Fluor 594 IgG and Alexa Fluor 488 IgG (Invitrogen, Waltham, MA) for 1 h. Fluoroshield Mounting Medium containing DAPI (Abcam, Cambridge, MA) was used to detect nuclei. Sections were analyzed using a confocal laser-scanning microscope (Zeiss) and the fluorescence intensity^{63 (link)} was analyzed and quantified by ImageJ (NIH, Bethesda, MD). We performed parallel immunohistofluorescence staining for all slides. We selected ATII cells to analyze fluorescence intensity of protein of interests by applying ImageJ and NIH Image 1.62 software. Identical image acquisition settings and exposure times were used. We calculated the corrected total cell fluorescence (CTCF) using the following formula: CTCF = Integrated Density − (Area of selected cell X Mean fluorescence of background readings).