In a target pH between 7.20 and 7.30 [15] and fixed at various time
In a target pH between 7.20 and 7.30 [15] and fixed at various time intervals. Day of sacrifice was considered Day 0, 18 hours after hCG injection, Day 0.5 (29.5hrs), Day 1 (42hrs), Day 3 (90hrs), Day 5 (138hrs).Western blot analysis, immunofluorescence, immunohistochemistry (IHC), and confocal microscopyMethodsCell lines, tissue, and gametesHuman and mouse tissue and gamete use was approved through Mayo Clinic Internal Review Board (IRB) and Institutional Animal Care and Use Committee (IACUC), respectively. GC-1spg and GC-2spd(ts) cell lines were obtained from American Type Culture Collection and maintained according to their recommendations. GC-1spg cells were originally generated by immortalizing mouse Type B spermatogonia with SV40 that demonstrate characteristics of a stage between type B spermatogonia and primary spermatocytes [11]. GC-2spd(ts) cells were first established by transfecting mouse spermatocytes with SV40 large T antigen [12, 13]. HP1 (Cbx3) knockdown was achieved byWestern blot, immunofluorescence and confocal microscopy were all performed as previously described [9, 10]. Formalin-fixed mouse testis MLN9708MedChemExpress Ixazomib citrate tissues were paraffinembedded and sectioned (5 m). Subsequently, IHC was performed as described [16]. Dilution of the P-Ser83-HP1 antibody [9, 10] was 1:50. Light microscopy slides were incubated with biotinylated goat anti-rabbit secondary antibody (Vector Labs), followed by HRP-streptavidin (Invitrogen), and immunoreactivity was monitored with Nova Red (Vector Labs). Negative control stains were done with incubation of secondary antibody only. Sections were counterstained with hematoxylin solution.Leonard et al. BMC Developmental Biology (2015) 15:Page 3 ofFluorescence images were obtained using confocal microscopy at 40X magnification using a Zeiss LSM-780 confocal microscope and images were analyzed using ZEN software. Light microscopy images were obtained at 40X magnification using a Zeiss AxioPlan2 with AxioVision software.Immunoelectron microscopySpermatozoa were processed for immunolabeling using a BioWave laboratory microwave (Ted Pella, Inc). Isolated spermatozoa were suspended in 1 agar and fixed in 4 paraformaldehyde + 0.1 glutaraldehyde, dehydrated in a series of ethanol, and embedded in LR White resin. Sections (0.1 M) were mounted onto Ni mesh grids for immunolabeling. Grids were hydrated on drops of phosphate buffered saline (PBS) + 0.1 TWEEN 20 followed by incubation with antigen retrieval solution (modified citrate, pH 6.1, Dako North America Inc.). Primary antibodies were incubated at room temperature in PBS + 0.1 TWEEN 20 followed by secondary gold (10 nm) conjugate (BBI Research). Electron micrographs were acquired using a JEOL 1400 TEM operating at 80 kV (Jeol USA, Inc.).mRNA isolation and RT-PCRa p-value < 0.05 were used to select significantly altered PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27385778 genes. A threshold of log2 fold change between -2 and 2 and a p-value > 0.95 were used to identify unchanged genes. Data analysis, hierarchical clustering, and ontology were performed as previously described [10]. Selected probes and their fold changes were loaded into Ingenuity Pathways Analysis Software (IPA; Ingenuity Systems) for annotation and network enrichment/analysis. Semantic relationship analysis with IPA generated significant networks of well-characterized pathways with the satisfaction of Fisher’s Exact Test. A subset of genes was validated by qPCR (Additional file 1: Fig. S1) as previously described [10, 16].ResultsFunction of.
