Alexa 488 and 594 conjugated secondary antibodies

As described earlier [20 (link)], cardiomyocytes were allowed to adhere on chambered slides and fixed in 4% paraformaldehyde (PFA). Slides were washed with phosphate-buffered saline-Tween 20 (PBS-T) twice and permeabilized with 0.1% sodium citrate and 0.05% triton-X100 for 10 min at RT followed by 0.1% sodium borohydride treatment for 5 min at RT to quench autofluorescence. Slides were washed three times with PBS-T and incubated with 3% normal horse serum (NHS) for 1 h. Specific primary antibodies were used in co-localization of DDIT3 (Abcam, Cambridge, UK, cat #ab233121) together with iNOS, using Alexa 488, and 594 conjugated secondary antibodies (Invitrogen Life Technologies, Carlsbad, CA, USA). DAPI mounting media was used to locate nuclei. Slides were examined for co-expression of DDIT3/iNOS in Dox treated cardiomyocytes in the presence or absence of SNAP or L-NAME using the microscopic (Carl Zeiss., LSM 5 PASCAL, Jena, Germany) analysis setup attached with Zeiss AxioCam HRM camera HAL 100 lamp and quantified by using AxioVision 4.8 software.

Recombinant human TNF-α was obtained from Cayman (Ann Arbor, MI, USA). Lipofectamine 2000 and Trizol reagents were purchased from Invitrogen (Carlsbad, CA, USA). The nucleoprotein and cytoplasm-protein extraction kits and ready-to-use immunohistochemical SP kits were purchased from KeyGEN BioTECH (Nanjing, JiangSu, China). The SignalStain DAB substrate kit was purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies targeting AKT, β-catenin and all unconjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies targeting phospho-AKT (p-AKT) and E-cadherin were purchased from Epitomics (Burlingame, CA, USA). Anti-phospho-PKD2 antibodies (for the IHC Assay) were purchased from Sigma. Antibodies against N-cadherin, vimentin, Zo-1, phospho-GSK3β (p-GSK3β), PKD1, PKD2, PKD3, phospho-S6K1(p-S6K1), phospho-ERK1/2 (p-ERK1/2), β-actin and histones were obtained from Cell Signaling Technology. Antibodies targeting green fluorescent protein (GFP) were purchased from Abcam (Cambridge, MA, USA). Alexa-488- and 594-conjugated secondary antibodies were purchased from Molecular Probes (Invitrogen).

Paraffin-embedded spinal cord tissue was cut into serial 10 μm parasagittal sections with a paraffin-microtome and subsequently double-stained for collagen IV (M3F7, Developmental Studies Hybridoma Bank, 1:500) and von Willebrand factor (vWF, Dako, 1:500). The immunohistochemical staining of paraffin sections was started by deparaffinization procedures followed by a standard immunohistological staining protocol. Briefly, after washing with PBS and antigen retrieval with 0.05% protease XXIV for 8 min at 37°C, sections were blocked with 5% donkey serum for 1 h at room temperature. Primary antibodies were incubated overnight at 4°C and after washing with PBS, incubation with Alexa 488 and 594-conjugated secondary antibodies (Molecular Probes, 1:500, respectively) was performed for 1 h at room temperature. For reduction of autofluorescent background, sections were additionally stained with 0.3% Sudan Black dye (Fluka).

“The mouse brains were cut into 50 μm coronal sections with a vibratome (Leica 1000) and treated with 50 mM ammonium chloride (NH₄Cl) for 10 min at room temperature. The slices were pre-incubated for 1 h in 1% BSA with 0.3% Triton X-100 in 0.1 M PB. The primary antibodies were incubated overnight at 4°C: rabbit anti-BACE1 C-Terminal (485–501; 1:250, Calbiochem), rabbit anti-BACE2 (Ab2; 44–59; 1:250, Calbiochem), mouse anti-human-PHF-tau (1:250, Pierce Biotechnology), and rabbit anti-Hsc70 (Hsp73; 1:250, Assay Designs). Alexa 488- and 594-conjugated secondary antibodies (Molecular Probes) were used. The slices were observed by fluorescence microscopy (Olympus IX81), and the individual images for GFP, BACE1, BACE2, PHF and Hsc70 expression were analyzed using Image Scope Pro software (Media Cybernetics). Deconvolution was performed using Image Scope Pro software (Media Cybernetics) and Cell Software (Olympus)”.

Reagents were obtained from Sigma (St. Louis, MO, USA), Lipofectamine 3000 for siRNA transfection was from Invitrogen (Thermo Fisher Scientific, Carlsbad, CA, USA), and HilyMax for plasmid transfection was from Dojindo (Kamimashikigun, Kumamoto, Japan). All-in-One First-Strand cDNA Synthesis Kits and All-in-One qPCR Mix were from GeneCopoeia (Rockville, MD, USA), and miRNA real-time PCR (RT-PCR) kits were from Genepharma (Shanghai, China). Luciferase assay kits were from Promega (Madison, WI, USA) and ChIP-IT Express Enzymatic Chromatin Immunoprecipitation Kits were from Active Motif (Carlsbad, CA, USA). SB431542 was purchased from Calbiochem/EMD (La Jolla, CA, USA). Antibodies used for immunoblotting and immunoprecipitation (IP) assays from Cell Signaling Technology (Danvers, MA, USA), Smad3 antibody for chromatin IP and western blotting from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and Alexa 488- and 594-conjugated secondary antibodies from Molecular Probes (Invitrogen, Thermo Fisher Scientific). Antibodies targeting phosphorylated Smad3 at Ser213, HIF-1α and all unconjugated secondary antibodies were from Abclonal (Wuhan, China).

Cells adherent to glass coverslips were fixed with 4% (w/v) paraformaldehyde. The staining procedure has been described in^{64 (link)}. Anti-HA (3F10, Roche), anti-Myc (9B11, Cell Signalling), a polyclonal rabbit antibody against an N-terminal epitope of murine Dectin-1^{44 (link)} and anti-LAMP-2 (2D5)^{67 (link)} were employed as primary antibodies which were then visualised by Alexa 488- and 594-conjugated secondary antibodies (Molecular Probes). DAPI (4-,6-diamidino-2-phenylindole from Sigma-Aldrich) was added to the embedding medium. Images were recorded with Olympus FV1000 or Leica SP5 confocal laser scanning microscope. Processing of images was performed with Olympus Fluoview, ImageJ and Adobe Photoshop software.