Alexa fluor 555 conjugated secondary antibody

Metformin-treated chondrocytes were seeded into on 15 mm cell slides (Nest Biotechnology) in 6-well plates. Cells were fixed with 4% paraformaldehyde (Beyotime, China) for 15 min and permeabilized with 0.1% Trion X-100 in PBS for 20 min. Chondrocytes were blocked with 10% BSA for 30 min and then incubated with anti-SIRT1 primary antibody (1:100, #8469, Cell Signaling Tech) overnight at 4 °C. Subsequently, cells were washed and incubated with Alexa Fluor 555-conjugated secondary antibody (1:500, A32727, Invitrogen) for 1 h at room temperature. Finally, nuclei were stained with DAPI in the dark, and fluorescence images were obtained using Fluorescence microscope (IX70, Olympus, Japan).

Kidneys were fixed in 4% paraformaldehyde (24 hours) and embedded in paraffin, and then, 5‐μm sections were subject to PAS and Masson's trichrome staining as per standard protocols. At least 10 random fields from each sample were analysed. Immunohistochemical staining was performed with anti‐C1orf54 (Sigma‐Aldrich, St. Louis, USA) as described,9 and C1orf54‐positive cells were counted under a microscope (Olympus, 400× magnification).
For immunofluorescence analysis, tissue sections were boiled in citrate buffer solution (10 minutes), treated with 0.2% Triton X‐100 (30 minutes, room temperature) and incubated overnight with rabbit anti‐C1orf54 (H00079630‐B04P, Sigma‐Aldrich) and rabbit monoclonal anti‐Ki‐67 (9129s, Cell Signaling Technology) to detect C1orf54 and proliferation, respectively. Villin antibody (Santa Cruz) was used to colocalize the C1orf54 and renal tubular. All sections were incubated (1 hour) with donkey anti‐rabbit Alexa Fluor^® 488‐conjugated secondary antibody (A‐21206, Invitrogen, Carlsbad, CA, USA) and donkey antimouse Alexa Fluor^®‐555 conjugated secondary antibody (A‐31570, Invitrogen), costained with DAPI and imaged under an Olympus microscope.

pMet-Ras^V12 S2 cells diluted in Schneider medium (to a concentration of 1×10⁶ cells/ml) were distributed in 96 well clear plates (Corning) containing 5 µl dsRNA aliquots at a concentration of ∼200 ng/µl. Plates were placed in plastic containers to reduce evaporation and incubated at 27°C for four days. Ras^V12 expression was induced by adding 0.7 mM CuSO₄ to medium 24 h prior to fixation. Cells were resuspended and transferred to concanavalin A coated plates and allowed to settle and adhere for 1 h. Cells were then fixed in 4% paraformaldehyde/PBS, washed, and blocked in 0.2% Triton X-100/0.2% BSA/PBS (PBT/BSA) and incubated overnight with an anti-pMAPK antibody (1/2,000; Sigma number M8159), washed in PBT/BSA, and revealed using an anti-mouse Alexa Fluor 555-conjugated secondary antibody (1/1,000; Invitrogen number A-21424). DAPI (0.04 µg/ml) was used to stain nuclei. Mowiol (9.6% PVA, Fluka) was added to wells prior to imaging. An automated fluorescence microscopy system (Zeiss Axiovert) was employed for plate imaging. Autofocus, image acquisition, and analysis were conducted using MetaMorph (Molecular Devices) software. The cell-scoring application in MetaMorph was used for cell segmentation and quantification of fluorescent signal.

On reaching 90% confluency, C2C12 cells were incubated in DMEM, supplemented with 2% horse serum (differentiation medium, DM) containing various concentrations of lumican. To block signal transduction pathways, cells were pre-treated with SB203580 (p38 inhibitor, Sigma-Aldrich, St. Louis, MO, USA), TC-I-15 (α2β1 integrin inhibitor, R&D Systems), echistatin (ανβ3 integrin inhibitor, R&D Systems), or integrin α7 neutralizing antibody (Origene, Rockville, MD, USA). After 3 days, the cells were fixed in 4% paraformaldehyde for 10 min, permeabilized in 10 mM sodium citrate buffer containing 0.1% Triton X-100 for 10 min, and blocked with 2% BSA for 1 h. Subsequently, cells were probed overnight with an anti- myosin heavy chain (MyHC) antibody at 4 °C. The cells were incubated with Alexa Fluor 555-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 h, and nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) for 10 min. Fluorescence images were obtained using an Axio Imager microscope (Carl Zeiss, Oberkochen, Germany) at 100× magnification. Area of MyHC⁺ myotubes and number of nuclei in MyHC⁺ myotubes were quantified in six randomly selected fields per group. Fusion index (%) was calculated as follows: 100 × number of nuclei in myotubes with two or more nuclei/total number of nuclei.

HCT116 cells were co-transfected with Flag-β-TrCP and His-CELF6 mutants for 48 h, cells were fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.5% Triton X-100 in PBS for 5 min. Fixed samples were blocked with 3% BSA/PBS, followed by incubation with anti-His (Proteintech, 66005-1-Ig) and Alexa Fluor 488 conjugated secondary antibody (Invitrogen). The stained samples were then incubated with anti-Flag (Proteintech, 66008-3-Ig) and Alexa Fluor 555 conjugated secondary antibody (Invitrogen). Images were captured using an Olympus FV1000 confocal microscope (Olympus, Japan).
For immunohistochemistry, paraffin-embedded cancerous colon tissues (n = 30) and normal colon tissues (n = 4) were purchased from Shanghai Outdo Biotech Co Ltd. The paraffin-embedded slide was deparaffinized, rehydrated, and blocked in sheep serum for 30 min, followed by incubation with anti-CELF6 (1:100, Abcam, ab173282) overnight at 4 °C. The slide was mounted with D.P.X. for histology analysis.

Dissected brains were fixed in 4% paraformaldehyde at 4°C overnight. For immunostaining using anti-Aβ antibody 4G8 (Covance) or rabbit polyclonal anti-Aβ_1–42 antibody ab12267 (AbCam), brains were pre-treated with 70% FA for 10 min. For immunostaining using aggregated-Aβ-specific antibody 7A1a (New England Rare Reagents), FA pre-treatment was not included. Primary antibody immunostaining was detected using Alexa Fluor-555-conjugated secondary antibody (Invitrogen). For immunostaining with anti-Rab5 antibody (AbCam), the secondary antibody used for detection was Alexa Fluor-647-conjugated goat anti-rabbit IgG (Invitrogen). Samples were observed and images collected using confocal microscopy (Zeiss LSM 510).