ICL: Rostral medulla, K and L are details from I and J, respectively. C57BL/6J male mice. Male offspring transporting a maternally inherited are phenotypically normal and were bred to C57BL/6J females to produce experimental embryos and offspring. In these litters, one-half of the mice are wild type and one-half carry a paternally inherited necdin deficiency and are functionally null. The timing of pregnancies was decided from the appearance of sperm plugs in the breeding cages, and the embryonic age of mice was confirmed by measuring crown-rump length.19 Identification of mutant offspring was performed by histochemical detection of -galactosidase activity and by Gatifloxacin polymerase chain reaction genotyping of snap-frozen tissue with oligonucleotide primers (LACZ1942F, 5GTGTCGTTGCTGCATAAACC; and LACZ2406R, 5TCGT-CTGCTCATCCATGACC). Animal Handling for Anatomical Studies Fetal mice were delivered from timed pregnant animals anesthetized with halothane (1.5% delivered in 95% O2 and 5% CO2) and transcardially perfused with 4% paraformaldehyde or 4% paraformaldehyde-2.5% glutaraldehyde in phosphate buffer at pH 7.2. Brainstems, spinal cords, and diaphragms were dissected out and postfixed in the same fixative answer; tissue was embedded in agar and cut on a vibratome (VT1000S; Leica, Germany) for single- and double-labeling immunohistochemistry. Immunohistochemistry To examine the detailed neuroanatomy of mice, we analyzed brainstem and spinal cord distribution of several anatomical and neuronal markers (observe Table 1 for antibodies used and their recommendations,20C31) from embryonic day (E)10 to E18. Mutant and wild-type mice within the same litter were processed together Gatifloxacin for comparisons. Transverse and sagittal sections (50 m) were serially collected in phosphate-buffered saline (PBS) and immunoreacted according to the following protocol. Free-floating sections were incubated with 1.0% bovine serum albumin (BSA; Sigma Chemical Co., St. Louis, MO) and 0.2 to 0.3% Triton X-100 in PBS for 60 minutes to reduce nonspecific staining and to increase antibody penetration. Sections were incubated overnight with main antibodies diluted in PBS made up of 0.1% BSA and 0.2 to 0.3% Triton X-100. The following day, sections were washed in PBS and incubated with specific secondary antibodies diluted in PBS and 0.1% BSA for 2 hours (biotin-, Cy3-, Cy5- or Cy2-conjugated donkey anti-rabbit, donkey anti-goat, donkey anti-rat, donkey anti-mouse IgG or donkey anti-mouse IgM; 1:200; all purchased from Jackson ImmunoResearch, West Grove, PA). Sections were further washed in PBS and those immunoreacted with fluorescent-conjugated secondary antibodies were mounted and coverslipped with Fluorsave mounting medium (Calbiochem, La Jolla, CA). In some experiments, sections were counterstained with Hoechst 33342 (Molecular Probes, Eugene, OR). Gatifloxacin When biotin-conjugated secondary antibodies were used, sections were labeled using a peroxidase method. After washes KRT17 in PBS, sections were incubated with standard Gatifloxacin peroxidase-conjugated ABC kit (1:100; Vector Laboratories, Burlingame, CA) for a further 2 hours. The reaction was detected with 0.08% diaminobenzidine (DAB) and 0.007% H2O2 in Tris-HCl buffer. Adjacent sections were counterstained with thionine (1%) to visualize tissue cytoarchitecture. DAB-immunostained sections were analyzed with an Olympus BX40 microscope and images were taken with a SPOTdigital Microscope video camera (Carsen, Markham, ON) connected to a computer running Image-Pro-Plus software (Media Cybernetics Inc., Silver Spring, MD). Acquired images were exported in TIFF format, and brightness and contrast adjusted in Adobe Photoshop 7.0. Table 1 Antibodies mice to wild-type littermates were applied to determine statistical significance at 0.05. Electron Microscopy For electron microscopy immunohistochemistry, 50-m free-floating sections were processed with pre-embedding immunoperoxidase. Sections were permeabilized by freeze-thawing at ?80C,32 rinsed in PBS, treated with 1% H2O2 in PBS, and incubated with 1.0% BSA to mask nonspecific absorption sites. Sections were incubated with the primary anti-neurofilament (NF) antibody, biotinylated secondary antibody (DAM), and ABC complex as specified above. Peroxidase staining was obtained by incubating the sections in 0.048% DAB, 0.024% CoCl2, 0.019% NAS, and 0.003% H2O2 in 0.1 mol/L phosphate buffer. All sections were extensively washed, osmicated in 1% OsO4 in 0.1 mol/L phosphate buffer for 20 minutes, dehydrated in ethanol, and flat-embedded in TAAB812 Epon. Thin sections were counterstained with uranyl acetate and lead Gatifloxacin citrate and examined using a Philips 410 transmission electron microscope. Brain Stem-Spinal Cord and Medullary Slice Preparations Fetal mice (E18) were decerebrated and the brain stem-spinal cord with or without the ribcage and diaphragm muscle mass attached was dissected following procedures much like those established previously.33,34 The neuraxis was continuously perfused at 27 1C (perfusion rate, 5 ml/minute; chamber volume, 1.5 ml) with mock cerebral spinal fluid that contained (mmol/L): 128 NaCl, 3.0 KCl, 1.5 CaCl2, 1.0 MgSO4, 24 NaHCO3, 0.5 NaH2PO4, and 30 d-glucose equilibrated with 95% O2-5% CO2. Details of the medullary slice preparation have been previously explained.35 Briefly, the brain stem was sectioned serially using a Leica vibratome in the transverse plane starting from the rostral medulla to within 150 m of the rostral boundary of.