Alexa fluor 594 donkey anti rat igg

Mouse embryonic kidney rudiments were fixed overnight in 4% PFA at 4 °C, embedded in gelatin for cryosection or in wax for paraffin section. Cryo-samples and paraffin samples were sectioned at 8 μm and 10 μm intervals, respectively. The sections were
blocked and penetrated for 1 h at room temperature in 3% FBS, 10% blood serum, and 0.2% triton-X100 followed by primary antibodies incubation overnight at 4 °C. Sections were immunostained using the following antibodies: CALB1 (1:400, C9848, Sigma), SIX2 (1:200, 11562-1-AP, Proteintech), GFP (1:200, ab6556, Abcam), Integrin α8 (1:400, AF4076, R&D), BrdU (1:100, #555627, BD), ETV5 (1:200, 13011-1-AP, Proteintech), phosphorylated-ERK (1:100, #4370, CST), cleaved-Caspase3 (1:200, #9661, CST), EphrinB1 (1:400, AF473, R&D), pFAK (1:300, #611722, BD), act Integrin β1 (1:100, #553715, BD) and N-cad (1:300, #610920, BD), Alexa Fluor 568 donkey anti mouse IgG (1:500, Invitrogen, A10037), Alexa Fluor 488 donkey anti rabbit IgG (1:500, Invitrogen, A32790), Alexa Fluor 594 donkey anti goat IgG (1:500, Invitrogen, A32758), Alexa Fluor 647 donkey anti rabbit IgG (1:500, Invitrogen, A31573) and Alexa Fluor 594 donkey anti rat IgG (1:500, Invitrogen, A21209). Sections were mounted with anti-fade mountant with DAPI (Invitrogene, S36938) and imaged under an Olympus BX-53 microscope or Zeiss LSM 800 confocal microscope.

Hemangiogenesis was quantified by performing corneal whole-mount CD31 staining as previously described [8 (link)]. Briefly, following neovascularization, we obtained a corneal button that was excised 0.5mm posterior from the limbal vessel and rinsed in PBS, and then fixed in acetone for 10 min at-20°C. Subsequently, the corneas were washed thrice in PBS, blocked with 3% BSA in PBS for 48 h at room temperature, stained with a rat anti-mouse CD31 antibody (BD Biosciences, Franklin Lakes, NJ) and incubated at 4°C overnight. The corneas were again washed with PBS and stained with a secondary antibody (Alexa Fluor 594 donkey anti-rat IgG; Invitrogen) for 3 h at room temperature. The stained whole mounts of corneas were placed on glass slides and examined under a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan). ImageJ was used for image analysis. Neovascularization was quantified by setting a threshold level of CD31-positive fluorescence, above which only vessels were depicted. Neovascularization was quantified in a masked manner. The vascularization area was outlined by using the innermost vessel of the limbal arcade as the border, and the surface area of corneal neovascularization (vascularized area/total corneal area) was quantified.

Mice were anesthetized with chloral hydrate and perfused transcardially with ice-cold saline followed by perfusion with 4% paraformaldehyde 24 h after reperfusion. The brains were removed and post-fixed overnight for 24 h in paraformaldehyde; they were then coronally sectioned (30 μm) using a vibrating microtome (Leica, Wetzlar, Germany). The sections were incubated in PBS containing 0.5% Triton X-100 and 10% normal goat serum for 1 h at room temperature, following by incubation with rat monoclonal anti-BrdU (1:400; Abcam, Cambridge, United Kingdom) at 4°C overnight. After several PBS rinses, sections were incubated with Alexa Fluor 594 donkey anti-rat IgG (1:200; Invitrogen, Carlsbad, CA, United States).
An experimenter (L-cY) coded all slides from the experiments before quantitative analysis. All BrdU-labeled cells in the DG of injured hemisphere were counted in each section by another experimenter (D-qS) blinded to the study coding. The total number of BrdU-labeled cells per section was determined and multiplied by 10 to obtain the total number of cells per DG using fluorescence confocal microscopy (EX61; Olympus, Tokyo, Japan).

Mouse anti-CNPase, rabbit anti-Hes1, and rabbit anti-Hes5 antibodies were purchased from Abcam Corporation (Cambridge, UK). Anti-MBP antibodys were obtained from Millipore Corporation (Billerica, MA) for immune staining or ABcam (ab62631) for Western blot, and immobilon Western chemiluminescent HRP substrate were obtained from Millipore Corporation. Mouse anti-β-actin monoclonal antibody was purchased from CWBIO Corporation (Beijing, China). HRP-linked anti-rabbit and anti-mouse IgG antibodies were from Cell Signaling Corporation (Beverly, MA). Alexa Fluor 594 Donkey anti-rat IgG was from Invitrogen Corporation (San Diego, CA), and primers of Notch1, Notch2, Notch4, Hes1, and Hes5 were synthesized by TaKaRa Corporation (Seta, Japan). PrimeScript RT reagent Kit with gDNA Eraser, SYBR Premix Ex Taq (TliRNaseH Plus), and EASY Dilution were also from TaKaRa Corporation. Cuprizone (bis-cyclohexanoneoxalydihydrazone, CPZ) was purchased from Sigma Corporation (Ronkonkoma, NY). Quetiapine was a generous gift from Professor Li Xinmin, University of Manitoba, Canada. Halothane was obtained from Halocarbon Laboratories (Kinderkamack, NY). Biodentine dental cement was obtained from Septodont Corporation (Saint Maur des Fossés, France). DMSO was obtained from Sigma Corporation, and MW167, a γ-secretase II inhibitor, was purchased from Calbiochem Corporation (La Jolla, CA).

Retinal flat mounts were prepared, stained, and analyzed as described [32 (link)]. Primary antibodies included rat anti-CD11b, clone M1/70, BD Bioscience; rat anti-Ki67, clone SolA15, eBioscience; and anti-β3-tubulin, ThermoFisher. Secondary antibodies (Invitrogen) included Alexa Fluor 594 donkey anti-rat IgG; or biotinylated anti-rabbit IgG and Alexa Fluor 488/streptavidin; or biotinylated anti-rat IgG and Alexa Fluor 350/Streptavidin. Cell nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories). GFP and YFP were detected by their fluorescence. For cell quantification, 8 individual 0.19 mm² fields (4 central, 4 peripheral) per retina were examined. The total number of cells through the entire retina within a field or contained within the field of the indicated retinal cell layer was counted. Results expressed as a mean number of cells per field or total cells per retina which was calculated based on a retinal volume of 2.7 mm³.