Rabbit polyclonal anti laminin antibody

To detect recombinant vFGF expression, Aag2, BHK-21, and C6/36 cells were infected at an MOI of 5 PFU/cell. At designated time points, supernatants and cells were collected and subjected to immunoblotting. To detect vFGF expression in midguts, mosquitoes were dissected, and midguts were collected, washed with PBS, briefly sonicated, and subjected to immunoblotting. Protein samples were mixed with 2× PLB (Protein Loading Buffer: 0.25 M Tris-Cl, pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol and 0.02% bromophenol blue) and incubated at 100 °C for 5 min. Proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto PVDF membrane (MilliporeSigma, Burlington, MA, USA), and probed with one of the following primary antibodies: (i) mouse monoclonal anti-HA antibody (Covance, Princeton, NJ, USA); (ii) rabbit polyclonal anti-collagen IV antibody (Abcam, Cambridge, MA, USA); (iii) rabbit polyclonal anti-laminin antibody (Abcam); (iv) mouse monoclonal anti-β-actin antibody (Sigma-Aldrich), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Sigma-Aldrich). Blots were developed using the SuperSignal West Pico Chemiluminescent substrate (Pierce Biotechnology/Thermo Fisher Scientific) and exposed to X-ray films.

In all, 8 µm cryosections were dried and stained with Hematoxylin Phloxine Saffron (HPS) (Dako). For Von Kossa staining, cryosections were incubated 30 min at RT in 1% silver nitrate in the dark followed by 15 min incubation in UV chamber and nuclear fast red counterstain. All sections were analyzed using a Pannoramic Midi II slide scanner and Case Viewer software (3D HISTECH Ltd.).
For immunohistochemical staining, cryosections were fixed in 4% PFA 15 min, incubated in PBS-Triton 0.5% 15 min followed by 1 h in PBS-BSA 1% at RT. Muscle sections were incubated with rabbit polyclonal anti-laminin antibody (dilution 1/200, Abcam) or rabbit polyclonal anti-osteocalcin or anti-Collagen III antibody (dilution 1/100 and 1/200, Abcam) in PBS-BSA 1% overnight at 4 °C. Corresponding normal polyclonal rabbit IgG was used as negative control (Abcam). Sections were washed with PBS and incubated with secondary antibody Donkey anti-rabbit Alexa Fluor 594 or Goat anti-rabbit Alexa Fluor 488 (1/500, Thermo) in PBS-BSA 1% for 1 h à room temperature. Slides were finally mounted in Vectashield antifade mounting medium with DAPI (Vector). All sections were analyzed using inverted epifluorescence microscope (DMi8, Leica) and Fiji software (ImageJ)