Goat anti rabbit cy3

The lumbar spinal cord segments were harvested 4 weeks after the fat grafts or vehicle treatments. The fresh-frozen sections were performed through the same method as the previously mentioned immunohistochemistry procedure [4 (link)]. For double immunofluorescence staining, the spinal cord dorsal horn was incubated with a mix of polyclonal p-IκB (1:100 dilution, Cell Signaling) and monoclonal NeuN (a neuron cell marker, 1:1000, Millipore, Temecula, CA); polyclonal p-NFκB (1:100 dilution, Cell Signaling, Boston, MA); monoclonal GFAP (an astrocyte marker, 1:1000 dilution, BD Biosciences San Diego, CA); and p-JNK (1:100 dilution, Cell Signaling, Boston, MA) overnight at 4°C. The appropriate secondary antibody conjugated with goat anti-rabbit Cy3 (red, Millipore, Temecula, CA) and goat anti-mouse Alexa Flour 488 (green, Invitrogen, Carlsbad, CA) was added. Images were acquired using a fluorescence microscope (Leica DMI6000).

Microspheres consisting of 1:1 collagen and alginate of approximately 30-200 µm in diameter were centrifuged, the supernatant was removed and 100 µl of the spheres themselves were injected subcutaneously into 12, approximately 24-week-old, female C57Bl/6 mice, in a total of three technical replicates, using G27 needles on a 1 ml syringe. Five days after injection the mice were killed and the hydrogel microsphere plaques were excised and fixed in 4% PFA at 4°C overnight. Plaques were stained according to a previously described protocol with an anti-mouseF4/80 antibody (1:200, Clone CI:A3-1, AbD Serotec, Bio-Rad, Cat#: MCA497GA) to visualize macrophages, or an anti-mouseLy6G antibody (1:200, Clone RB6-8C5, Abcam, Cat#: ab25377) to visualize neutrophils, coupled to secondary goat-anti-rat-Cy5 (Millipore) or goat-anti-rabbit-Cy3 (Millipore) antibodies. The antibodies have been validated in the scientific literature as well as by the manufacturers. Animal studies were approved by the North Stockholm Research Animal Ethical Council.

The rats were euthanized, and subsequently the ventral horn areas of spinal cord segments (L3–5) were collected on W1 and postfixed overnight in 4% paraformaldehyde in 0.1 M phosphate-buffered saline at 4°C before being transferred into a 30% sucrose solution. To detect microglia activation, the sample sections were double-labeled for phosphorylated p38 mitogen-activated protein kinase (MAPK) (pp38 MAPK, 1:200; Cell Signaling) and oxycocin-42 (OX-42, microglia marker, 1:200; Serotec). To detect the anti-inflammation reaction in neurons, the sample sections were incubated with a mix of iNOS (1:200, Abcam), COX-2 (1:200, Cell Signaling), and monoclonal NeuN (neuron cell marker, 1:1000; Millipore, Temecula, CA, USA). To examine the role of autophagy, antimicrotubule-associated protein light chain-3 (LC3B) rabbit polyclonal antibody (1:200, Cell Signaling), autophagy protein 5 (ATG5, 1:200; Thermo), and monoclonal NeuN (1:1000; Millipore) were used. An appropriate secondary antibody conjugated with goat antirabbit Cy3 (red; Millipore) and goat antimouse Alexa Flour 488 (green, Invitrogen, Carlsbad, CA, USA) was added. Images were acquired using a fluorescence microscope (Leica DM 16000).

Tissue for immunohistochemistry was obtained at day 5 of wound healing experiments. A biopsy punch was used to obtain wound areas, which were frozen and cut in OCT compound (Tissue Tek, Sakura).
Cryosections were fixed with 4 % PFA. Unspecific binding sites were blocked with Avidin/Biotin‐Blocking‐Kit (Vector Laboratories) and with protein blocking reagent (ROTI®ImmunoBlock, Roth) supplemented with 5 % BSA (PAN‐Biotech) and 20 % goat serum (Vector Laboratories). The slides were subsequently permeabilized with 0.1 % triton X and were incubated with primary antibodies against F4/80 (BM8, BioLegend), Cd68 (Polyclonal, abcam), iNos (Polyclonal, abcam), Cd163 (TNKUPJ, Thermofisher), Cd206 (polyclonal, abcam) and Arginase‐1 (polyclonal, Novus Biologicals). Goat ant‐rabbit AF488 (Invitrogen), goat anti‐rat biotin followed by streptavidin‐Cy3 (both BioLegend), goat anti rat AF488 (abcam) and goat anti‐rabbit Cy3 (Millipore) were used for detection. Nuclei were stained with Hoechst (Life Technologies). Analyses were performed with fluorescence microscopy (Leica DM6000B) and with confocal microscopy (Leica SP8).

Larvae were synchronized as described (Stiernagle, 2006 ) and heat shocked at the desired stage in M9 buffer at 33 °C for 15 min using a thermal cycler. Anti-ELT-2 antibody was a gift of J. McGhee (University of Calgary, Canada). Anti-PHA-4 antibody was a gift of S. Mango (Harvard, MA). Cy3 goat anti-mouse and Cy3 goat anti-rabbit was obtained from Sigma. Fixation and permeabilization of L2-adult (Finney and Ruvkun, 1990 (link)) or L1 (Sommermann et al., 2010 (link)) stage worms was carried out as described.