Af594 donkey anti rabbit

The middle part of gastrocnemius muscle and distal end of crush nerve were harvested and then cryosectioned into 8-μm and mounted on Superfrost/Plus slides (Menzel-Glaser, Braunschweig, Germany). The tissue slices were subjected to immunohistochemistry with antibodies against von Willebrand factor (1:200 dilution, Santa Cruz Biotechnology, Dallas, TX, USA), isolectin B4 (1:200 dilution, Vector Laboratories, Burlingame, CA, USA), CD 68 (1:200 dilution, Bio-rad, Hercules, CA, USA), neurofilament (1:200 dilution, Merck Millipore, Burlington, MA, USA), S-100 (1:200 dilution, Merck Millipore, Burlington, MA, USA), desmin (1:200 dilution, Abcam, Cambridge, MA, USA), and acetylcholine receptor (1:200 dilution, Merck Millipore, Burlington, MA, USA), for detection of nerve and muscle regeneration/degeneration. The immunoreactive signals were observed using AF 488 donkey anti–mouse IgG and AF594 donkey anti-rabbit (1:200 dilution, Invitrogen, Carlsbad, CA, USA) under a confocal microscope [29 (link)].

Cells were fixed with 4% formalin after 7 days of chondrogenic differentiation or after 1 or 3 days of subsequent osteogenic differentiation. Cells were blocked with 4% bovine serum albumin (Sigma) and immunofluorescent staining was performed using the primary antibodies rabbit anti-Col2a1 (Rockland, 1:100) and goat anti-Col1a1 (Santa Cruz, 1:100) overnight at 4 °C and secondary antibodies AF594 donkey anti-rabbit (Invitrogen, 1:250) and biotinylated donkey anti-goat antibody (Santa Cruz, 1:200) for one hour at room temperature (RT), respectively, followed by incubation with FITC-Streptavidin (Biolegend, 1:200) for 30 min at RT. Host-specific IgG isotype controls were used as a negative control instead of primary antibodies.

Sciatic nerve and gastrocnemius muscle were subjected to immunohistochemistry with antibodies against NT-3 (1:200, Millipore), BDNF (1:200, Abcam), CNTF (1:200, Abbiotec), GDNF (1:200, Abbiotec), S-100 (1:200, Serotec), neurofilament (1:200, Millipore), desmin (1:200, Proteintech), acetylcholine receptor (1:200, Millipore) and 8-oxo-dG (1:500, R&D) for detection of nerve and muscle regeneration. AF 488 donkey anti-mouse IgG and AF594 donkey anti-rabbit (Invitrogen; 1:200 dilutions) were used to stain and viewed by confocal microscopy capture image. Five consecutive resections (approximately maximum diameter) were chosen and measured by the IS 1000 image analysis system according to our previous study [31 (link)]. The number of cells was counted in randomly selected 20 squares from100 squares in an ocular grid. The region of maximum diameter of the resected nerve tissue and muscle was chosen to be examined. Areas of activities (0.2 mm²) appeared dense against background and were measured.

The slides were permeabilized and blocked with 10% donkey serum, 2% BSA, 0.05% tween in PBS for 1 hr at room temperature. For lung cells, the slides were incubated with primary antibodies anti-CD31 (BD Pharmingen, Cat#: 550274, 1:25) and anti-RAGE (Abcam, Cat#: Ab3611, 1:3200) at 4°C overnight. The brain ECs were incubated with primary antibodies anti-CD31 (BD Pharmingen, Cat#: 550274, 1:25) and anti-PTN (Santa Cruz Biotechnology, Cat#: sc-74443, 1:3200) at 4°C overnight. For the heart samples, primary antibodies anti-AQP7 (Novus Biologicals, Cat#: NBP1-30862, 1:3200) and anti-CD31 (BD Pharmingen, Cat#: 550274, 1:25) were used and incubated at 4°C overnight. The next day, slides were washed and incubated with the fluorescence-conjugated secondary antibody (AF488 donkey anti-rat 1:300, Invitrogen Cat#: A-21208; AF594 donkey anti-rabbit 1:300, Invitrogen Cat#: A-21207; AF594 goat anti-mouse 1:300, Invitrogen Cat#: A11032), followed by washing with 1x PBS. Cells were stained with DAPI and mounted on ProLong Gold mounting medium (Invitrogen, Cat#: P36934). Images were taken with a confocal microscope LSM880 (Zeiss) and analyzed by Zen software (Zeiss).

Immunohistochemistry was performed as described previously (Richardson et al., 2012). 10 μm sections were used for all studies. Primary antibodies used: rat ant‐p21 (HUGO291, Abcam ab107099), goat‐anti‐troponin C (Abcam, ab30807), rabbit anti‐p16 (Rockland, 100‐401‐170) and rat anti‐CD31 (MEC13.3, BD Biosciences, 550274). Secondary antibodies used were donkey anti‐rat AF594 (Life Technologies, A21209), donkey anti‐goat AF 488 nm (Life Technologies, A11055), donkey anti‐rabbit AF594 (Life Technologies, R37119), donkey anti‐goat AF488 (Life Technologies, A11055) and donkey anti‐mouse AF647 (A31571, Life Technologies). Slides were mounted in Vectarshield containing DAPI (Sigma, MBD0015). 5‐ethynyl‐2‐deoxyuridine (EdU) labeling performed with Invitrogen Click‐iT EdU Alexa Fluor 594 Imaging Kit (Life Technologies, C10339).