Alexa fluor 488 conjugated affinipure donkey anti mouse igg

Orbital fibroblasts (1.5 × 10⁵ cells/ml) without or with macrophages (2.25 × 10⁵ cells/ml) were cultured in free-floating gels for 4 days, and processed for immunofluorescence using Rhodamine-labeled Phalloidin (Molecular Probes, OR, USA), anti-αSMA primary antibody (Sigma-Aldrich, UK) and Alexa Fluor 488-Conjugated AffiniPure donkey anti-mouse IgG (Jackson Immunoresearch, UK) as previously described^{59 (link)}. Gels were mounted with Fluoroshield mounting medium with DAPI (abcam, UK), and imaged using a Nikon Eclipse Ti microscope with 10X Plan Fluor 0.3NA or 40X S Plan Fluor ELWD 0.60NA objectives. For quantification, full stacks (50 μm in Z stack with 1 μm step) of 5–6 fields were acquired using the 10X objective and processed using the NIKON NIS Element Software. Maximal intensity projections were generated and cells manually traced to get outlines as selected areas for calculations. Background signal was subtracted before calculating fluorescence intensity under TRITC or FITC channels. The fluorescence intensities of TRITC and FITC were then divided by the selected area to obtain fluorescence intensity per unit area. For representative single cell images, cells were imaged with 40x objective, processed by deconvolution, and shown as maximal intensity projections.

Slides of paraffin-embedded primary HCC tissues were used for immunochemistry and stained with antibodies against Prp19 and Cdc5L. The exact procedure and the semi-quantitative method were reported elsewhere [27 (link)]. Images were processed with a Nikon microscope with NIS Element F3.2 software (Nikon, Melville, NY, USA).
When subjected to immunofluorescence assay, unfixed frozen sections were generally processed as described below, briefly. First, sections were incubated with rabbit anti-human Prp19 polyclonal antibody (1:250) and mouse anti-human Cdc5L Monoclonal antibody (1:20) overnight at 4 °C after blocked by Phosphate buffered saline (PBS) containing 5% Bovine serum albumin (BSA). They were then washed by PBS carefully. These sections were incubated with Alexa Fluor^® 647-conjugated AffiniPure Donkey Anti-Rabbit IgG and Alexa Fluor^® 488-conjugated AffiniPure Donkey Anti-Mouse IgG (both 1:500) at room temperature for 2 h (Jackson ImmunoResearch Inc., West Grove, PA, USA). Finally, sections were added with Mounting Medium for Flurescence with DAPI and detected by Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany).

Primary antibodies were: mouse anti-human HSP70, specific for stress-inducible HSPA1A (StressMarq, Canada, SMC-100B; 1:100 ICC; 1:1000 WB), mouse anti-Flag M2 (Sigma-Aldrich, #F1804; 1:400 ICC); rabbit anti-FUS (Proteintech, USA, 11570-1-AP; 1:400 ICC), mouse anti-human SOD1 (Sigma-Aldrich Canada, SD-G6; 1:100 ICC), rabbit anti-acetyl-histone H3K9/K14 (Cell Signaling, #9677; 1:400 ICC; 1:1000 WB), mouse anti-GAPDH (MediMabs, Canada, #MM-0163; 1:1000 WB), moue monoclonal anti-acetylated tubulin (Sigma-Aldrich #T6793; 1:1000 WB) and rabbit anti-α-tubulin (Abcam #ab15246; 1:1000 WB).
Secondary antibodies (Jackson Immunoresearch: Cedarlane, Canada): Alexa Fluor 488-conjugated Affinipure donkey anti-mouse IgG (1:300); Cy3-conjugated donkey anti-mouse IgG 1:300); Cy5-conjugated donkey anti-rabbit IgG (1:300), and HRP-conjugated goat anti-mouse (1/5000 for WB, 1/500 for IC) or donkey anti-rabbit IgG (Jackson Immunoresearch (1:2500).
HDAC inhibitors and HSP-inducing drugs: SAHA (suberoylanilide hydroxamic acid) and tubastatin A (Cayman Chemical: Cedarlane, Canada); tacedinaline, RGFP109 and RGFP966 (Selleckchem: Cedarlane, Canada); sodium phenylbutyrate (Selleckchem); arimoclomol (Toronto Research Chemicals, Canada); NXD30001 was previously supplied by NexGenix Pharmaceuticals (Cha et al. 2014 (link)).