Alexa fluor 488 conjugated donkey anti rabbit igg secondary antibody

To confirm PDH and PDK2 levels, we performed PDH and PDK2 staining with primary antibodies for each. Immunohistochemistry with PDH and PDK2 antibodies (Abcam, Cambridge, UK) was performed. Brain tissues were incubated in monoclonal rabbit antibody to rat PDH (diluted 1:100, ab168379) and a monoclonal rabbit antibody to rat PDK2 (diluted 1:100, ab68164) overnight in a 4 °C incubator. After washing, the sections were immersed in secondary antibody (PDH: Alexa Fluor 594-conjugated donkey anti-rabbit IgG secondary antibody, PDK2: Alexa Fluor 488-conjugated donkey anti-rabbit IgG secondary antibody, respectively, both diluted 1:250; Molecular Probes, Invitrogen, Carlsbad, CA, USA) for 2 h at room temperature (RT). The brain tissues were placed on gelatin-coated slides for observation under a microscope. To check the intensity of fluorescence of PDH and PDK2, we used ImageJ (version 1.47c; NIH, Bethesda, MD, USA) and the following steps were executed: select 100 cells in one brain tissue and then click the menu option Analyze → Measurement. (magnification = 80×).

Astroglial cells were evaluated by the glial fibrillary acidic protein (GFAP) to confirm the effect of cerebrolysin on pilocarpine-induced seizure. Following brain cryostat sectioning, we stained the cut tissue. After precleaning to eliminate the remaining blood cells in the tissues, we put the tissues in monoclonal rabbit anti-GFAP antiserum (diluted 1:1,000, AbD Serotec, United Kingdom) and kept them overnight for 16 h at 4°C. Sixteen hours later, the tissues were placed in Alexa Fluor 488-conjugated donkey anti-rabbit IgG secondary antibody (diluted 1:250, Invitrogen, Grand Island, NY, United States) for 2 h at room temperature. Then, the samples were placed on the slides. The slides were dried and mounted with DPX (Sigma-Aldrich Co., St. Louis, MO, United States), and the tissues were observed through an Axioscope microscope. Astroglial cells were then quantified in the stratum oriens (SO), stratum pyramidale (SP), and stratum radiatum (SR) of hippocampal CA1 and CA3. Astroglial cells were expressed as their density (cell count/mm²).

To analyze microtubule damage from the brain sections, MAP2 was detected by immunofluorescence staining. MAP2 antibodies (Alpha Diagnostic Intl. Inc., San Antonio, TX, USA) for immunohistochemical staining were used as in a previous study [80 (link)]. Brain sections were soaked in a polyclonal rabbit anti-MAP2 serum (diluted 1:200, Alpha Diagnostic Intl. Inc., San Antonio, TX, USA) with PBS containing 0.3% TritonX-100 overnight in a 4 °C incubator. After overnight incubation, we washed the sections three times for 10 min with 0.01 M PBS, and then the brain sections were soaked in a solution of Alexa-Fluor-488-conjugated donkey anti-rabbit IgG secondary antibody (diluted 1:250, Invitrogen, Grand Island, NY, USA) for 2 h at RT. The brain sections were raised on gelatin-coated slides for analysis under a microscope. We used the Image J (NIH, Bethesda, Rockville, MD, USA) program to measure the microtubule damage and measured the mean gray value.

Hematoxylin and eosin (H and E) staining was performed in the liver tissue collected from Sam68^f/f and Sam68^LKO mice and fixed in 4% paraformaldehyde (PFA) by following the protocol reported by Dai et al. [51 (link)]. For assessing Sam68 expression, liver tissues were collected from HFD- or ND-fed mice, and cryosections were subjected to immunofluorescence staining. Briefly, the sections were blocked in 10% donkey serum in PBST at room temperature for 1 h, then incubated with Sam68 antibody (1:100) for overnight at 4 °C. Next day, the sections were washed with PBST, followed by incubation with Alexa Fluor 488-conjugated Donkey anti-rabbit IgG secondary antibody (Invitrogen, Waltham, MA, USA) (Green, 1:300) for 1 h at room temperature in dark. Nuclei were counterstained with DAPI (blue, Vectashield, Antifade Mounting Medium with DAPI). The fluorescence signal was examined under Nikon NI-S-E Microscope (Nikon America, Inc., Melville, NY, USA).