Cresyl violet

The primary antibody used for Met immunohistochemical study was mouse anti-Met (Met, B-2; sc-8057; lot No. C2807; Santa Cruz Biotechnology, Santa Cruz, CA; immunogen: peptide corresponding to amino acids 1330-1379 of mouse Met (NCBI# NP 032617)). Using immunoblotting methods, the antibody recognizes the recombinant Met protein (Santa Cruz), and a minor band at 170 kD and a major band at 140 kD in brain tissue homogenates (see below). These bands represent pre-processed and processed forms, respectively, of the Met receptor. Only the pre-processed band was detected in homogenates prepared from mouse neocortex in which the Met gene was deleted from the dorsal pallium (data not shown). A mouse monoclonal antibody, 1G9, generated in the laboratory against adult mouse hippocampal homogenates, cross-reacts specifically with phosphorylated neurofilament-H (NF-H) (Pennypacker et al., 1991 ), and was used to stain developing axons.
Postnatal mice were deeply anesthetized with sodium pentobarbital (60 mg/kg i.p.) prior to transcardial perfusion with room temperature phosphate-buffered 4% paraformaldehyde (pH 7.3) containing 1.3% L-lysine and 0.24% sodium periodate. After postfixation overnight at 4°C, brains were cryoprotected via sequential 12-hour incubations in 10%, 20%, and 30% sucrose in PBS, pH 7.5. Fetal brains were harvested and immersion-fixed overnight at 4°C prior to cryoprotection.
Fetal and P0 fixed brains were frozen in embedding medium (Triangle Biomedical Sciences; Durham, NC) over liquid nitrogen vapors and stored at -80°C until sectioned with a cryostat at 20 μM. Sections were collected on gelatin-subbed slides and stored at -80°C until processed. Fixed brains from P7 through P35 were frozen, cut at 40 μM with a sliding microtome (Leica, Bannockburn, IL) and free-floating sections were stored in a cryopreservative solution at -20°C until processed. One series of sections from selected brains were stained with Cresyl Violet as previously described (Hockfield, 1993 ).
For Met immunohistochemical processing, both cryostat and free-floating sections were rinsed in PBS and then incubated for 5 minutes in 0.5% H₂0₂ in PBS to quench endogenous peroxidases. The sections were then rinsed in PBS before 25 min incubation in 0.1 M Tris-glycine (pH 7.4). Several more PBS rinses preceded a 1.5 hr incubation in unlabeled donkey anti-mouse IgG (Fab; Jackson Immunoresearch, West Grove, PA) to block endogenous mouse immunoglobulins. Sections were further blocked in several rinses of Blotto-T (4% Carnation dried milk in PBS containing 0.2% Triton-X-100). Blocked sections were incubated in primary mouse anti-Met antibody diluted 1:250 in Blotto-T. Cryosections were incubated for 2-4 hours at room temperature; free-floating sections were incubated for 48-72 hours at 4°C. Following washes in Blotto-T, sections were incubated for 1 hour at room temperature in 1:1000 biotin-SP-conjugated donkey anti-mouse IgG (Jackson Immunoresearch) diluted in Blotto-T. Sections then were rinsed several times in PBS and processed by the ABC Elite histochemical method (Vector, Burlingame, CA). Met-specific antibody complexes were visualized by incubating the sections for 2-4 minutes at room temperature in 0.5% 3′3′-diaminobenzidine (DAB) with 0.015% H₂0₂. The stained sections were rinsed in PBS, and free-floating sections were mounted on gelatin coated slides. Finally, sections were dehydrated with ethanol, cleared with xylene, and coverslipped in DPX (Fisher, Pittsburgh, PA) for microscopic analysis. Phosphorylated NF-H staining was performed using a similar immunohistochemical protocol for free-floating sections, but with the following specific parameters: 1) The 1G9 primary antibody was diluted 1:200 in Blotto-T, 2) biotin —SP-conjugated donkey anti-mouse IgM secondary antibodies were diluted 1:1,000 in Blotto-T, and 3) antigen/antibody complexes were visualized using standard ABC reagents (Vector), followed by DAB histochemistry.