Alexa fluor 647 goat anti rabbit igg antibody

The cells were fixed with 100% methanol for 10 min at −20 °C and then washed thrice for 5 min with IF buffer solution (10 × stock: 38.0 g NaCl, 9.38 g Na₂HPO₄, 2.07 g NaH₂PO₄, 2.5 g NaN₃, 5.0 g BSA, 10 mL Triton X‐100, and 2.5 mL Tween‐20 in 500‐mL PBS), followed by treatment with a blocking solution (3% BSA in 1 × IF washing solution) for 30 min. The cells were then incubated for 1 h at 25 °C with mouse SMA antibody (1 : 400, ab7817; Abcam, Cambridge, UK), rabbit anti IL‐6 antibody (1 : 200, ab6672; Abcam), or rabbit anti GFP antibody (1 : 400, #598; Medical & Biological Laboratories, Nagoya, Japan) in 1 × IF buffer. After three washing cycles with 1 × IF buffer, the cells were incubated for 1 h with Alexa Fluor 488 Goat anti‐mouse IgG antibody (Invitrogen, Carlsbad, CA, USA), Alexa Fluor 568 Goat anti‐rabbit IgG antibody (Invitrogen), or Alexa Fluor 647 Goat anti‐rabbit IgG antibody (Invitrogen), respectively, diluted 1 : 400 in 1 × IF buffer solution. For counterstaining, the cell nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA). After three washing cycles with 1 × IF buffer, the slides were mounted and imaged using a fluorescence microscope [BZ‐710 (Keyence, Osaka, Japan) and ECLIPSE Ti2 (Nikon, Tokyo, Japan)].

The HEK293 cells transiently transfected with pcDNA5/FRT plasmid encoding NUDT8 gene were grown on a glass-bottom 96-well cell imaging plate for 24 h. After staining with 100 nM MitoTracker Red CMXRos for 1 h, the cells were fixed with 4% PFA in PBS for 15 min at 25 °C, followed by permeabilization with 0.3% Triton X-100 in PBS. The cells were washed with PBS and blocked with 3% BSA in PBS for 1 h at 25 °C and then incubated for 12 h at 4 °C with 1% BSA in PBS containing 2 μg ml⁻¹ rabbit primary polyclonal antibody Nudt8 (PA5-59493, 1:50, Thermo Fisher Scientific), followed by 1 h incubation with Alexa Fluor 647 goat anti-rabbit IgG antibody (A32728, 1:1,000, Invitrogen) and 1.0 μg ml⁻¹ Hoechst 33342 at 25 °C. The cells were washed three times with PBS before imaging.

Rats with diabetic ischemic wounds were randomly divided into a PBS control group, a BM-MSC topically treated group, and a BM-MSC IV-treated group. Topically treated rats received 1 × 10⁶ BM-MSCs in 100 μL of PBS intradermal injections at four injection sites around the wound immediately after creation [21 (link)]. In contrast, IV treated rats received 5 × 10⁶ BM-MSCs in 1000 μL PBS via the left femoral vein right immediately after wound creation [6 (link)]. To evaluate the distribution of MSCs in this model at days 3, 7, and 14, the mKate2-labeled BM-MSCs were applied in the same dosage and administration method as previously described to trace using fluorescence imaging and immunohistochemistry (IHC) analysis.
Rats were perfused with 60 ml of normal saline through the left ventricle followed by 100 ml of 4% paraformaldehyde. After perfusion, the major organs (Fig. 6a) were dissected and subjected to fluorescence imaging. Fluorescence imaging was performed using In Vivo Xtreme Imaging System (Bruker Corporation, Karlsruhe, Germany) with excitation wavelength 588 nm and emission wavelength 635 nm. For IHC analysis, the lung, pancreas, and wound sites were detected with antibodies specific against mKate2 (1:4000; Product # R10367; Invitrogen), the latter subsequently visualized with Alexa Fluor® 647 Goat Anti-Rabbit IgG antibody (1:500; Product # A-21245; Invitrogen).

After administration on day 14, wound and right gastrocnemius sections were incubated with anti-mKate2 rabbit polyclonal antibody (1:4000; Product # R10367; Invitrogen) and anti-CD31 mouse monoclonal antibody (1:1500; Product # ab24590; ABcam) overnight at + 4 °C, followed by a further incubation at room temperature for 1 h with Alexa Fluor® 647 goat anti-rabbit IgG antibody (1:500; Product # A-21245; Invitrogen) and Alexa Fluor® 488 goat anti-mouse IgG antibody (1:500; Product # ab150117; ABcam). Nuclei were counterstained with DAPI stain (Product # G1012; Servicebio).