Cy3 conjugated donkey anti mouse igg

For immunocytochemistry, AdMSCs and NRG1-AdMSCs were grown in four-well Lab-Tek II chamber slides (Nunc, USA) at a density of 2×10⁴ cells/mL. Briefly, the cells were fixed with 4% paraformaldehyde for 30 min at 4°C. The cells were incubated in blocking solution (10% normal donkey serum) for 30 min and then with rabbit polyclonal anti-NRG1 (1:200; Santa Cruz Biotechnology, USA) for 16 h at 4°C. After washing with PBS, the cells were incubated with Cy3-conjugated donkey anti-mouse IgG (1:500; Jackson ImmunoResearch, USA) for 1 h at room temperature (22 ± 3°C). After final washing with PBS, the cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI).
For immunohistochemical staining, the 40-μm-thick coronal sections were incubated with 10% normal donkey serum for 1 h and then with the following primary antibodies for 16 h at 4°C: rabbit polyclonal anti-NRG1 (1:200; Santa Cruz Biotechnology), mouse monoclonal anti-human nuclei (1:200; Millipore Corporation, USA). After washing with PBS, the sections were incubated with the following secondary antibodies for 2 h at room temperature (22 ± 3°C): Cy3-conjugated donkey anti-mouse IgG (1:500; Jackson ImmunoResearch), Alexa488-conjugated donkey anti-rabbit IgG (1:500; Jackson ImmunoResearch). Then, after washing with PBS, the sections were mounted and observed using a confocal microscopy (LSM 510 Meta; Carl Zeiss Co., Germany).

Standard protocol was followed to prepare samples for immunohistochemistry (Kango-Singh et al., 2002 (link)). Briefly, third instar larval brains were dissected in PBS and fixed for 20 min in 4% paraformaldehyde. The samples were washed in PBST (PBS+0.4% Triton-X-100), blocked in PBSTN (PBST + 2% Normal donkey serum) and incubated at 4^oC overnight in primary antibodies at appropriate dilution. The samples were then washed three times in PBST and processed for immunohistochemistry by incubation in appropriate secondary antibodies for 2 h, washed twice in PBST, mounted in VectaShield (Vector Labs), and scanned using Laser Scanning Confocal Microscopy (Olympus Fluoview 1000, 3000) at 20× magnification. The primary antibodies used were mouse anti-Prospero (DHSB, 1:100), rat anti-Miranda (Ab-Cam, 1:200), mouse anti-PH3 (1:250, DSHB), mouse anti-DIAP1 (1:100, gift of B. Hay), mouse anti-β-galactosidase (1:100, DHSB), rabbit anti-β-galactosidase (1:100, DHSB). Secondary antibody used were donkey Cy3-conjugated anti-mouse IgG (1:200, Jackson ImmunoResearch), donkey Cy3-conjugated anti-rabbit IgG (1:200, Jackson ImmunoResearch) and donkey Cy5-conjugated anti-rat IgG (1:250, Jackson ImmunoResearch).

Antibody staining was performed by using the following primary antibodies: mouse anti PH3 (1:200, Cell Signaling Technology), mouse anti DIAP1 (1:200, from Dr. Bruce Hay), rat anti ELAV (1:300, DSHB), mouse anti Armadillo (1:100, DSHB), mouse anti Fas2 (1:100, DSHB), rat anti E-Cad (1:100, DSHB), and mouse anti MMP1 (1:200, DSHB). The secondary antibodies used to detect primary antibodies were: Donkey Cy3 conjugated anti mouse IgG (1:200, Jackson ImmunoResearch) or Donkey Cy5 conjugated anti rat IgG (1:200, Jackson ImmunoResearch).
Immunohistochemistry was performed using standard protocol (Kango-Singh et al., 2002). Briefly, third instar larvae of appropriate genotypes were dissected in 1X PBS, fixed in 4% paraformaldehyde (PFA). The discs were incubated with appropriate primary (overnight incubation at 4°C), and secondary (2 hours at room temperature) antibodies. 1X PBST was used to permeabilize the tissue, and wash off unbound antibodies following each incubation. The processed tissue was mounted in Vectashield (Vector labs). A minimum of 15 discs were analyzed for each staining and genotype.