Phaseolus vulgaris leucoagglutinin pha l

Rhodamine labeled Phaseolus Vulgaris Leucoagglutinin (PHA-L) was purchased from Vector Labs (RL-1112). Lectin staining was performed as previously described (Blackburn and Lupashin, 2016 (link)) with minor modifications. Briefly, cells were cultured as indicated to 70–80% confluency on collagen-coated coverslips. Cells were washed with PBS and fixed in 1% PFA for 15 min, quenched with 50 mM NH₄Cl for 10 min, and washed with PBS, followed by a 30 min block with PBSB. The coverslips were incubated in PBSB containing 2 μg/mL PHA-L in a 4°C cold-room (kept in the dark during incubation) with gentle rocking for 30 min. Then, coverslips were stained with Hoechst 33342, washed with PBS three times, and mounted for imaging. The images were obtained using a Nikon NIS-Elements C confocal microscope with a 60× oil lens and Z-stacks at 0.3 μm intervals. Maximum intensity projections were processed and quantified using Nikon NIS-Elements analysis software. All images in the same experiment were captured and processed with the same setting.

The anterograde tracer, Phaseolus vulgaris leuco-agglutinin (PHA-L; Vector Laboratories, Burlingame, CA), was injected into the PB of five rats. Animals were anesthetized intraperitoneally with ketamine-xylazine and their head mounted in a stereotaxic apparatus as described above. Using the same stereotaxic coordinates as described in the retrograde tract tracing experiment section, 2.5% PHA-L in 0.01M PB at pH 8.0 was injected by a glass micropipette into the PB using iontophoresis (6 μA positive current, pulsed on-off at 7 s intervals) for 15–20 min. Following a 10 day transport time, animals were fasted for two days and then refed for 2 h. The weight of the animals was monitored during the recovery period. All of the animals gained weight during the recovery period compared to their preoperative weight. Perfusion of the animals with fixative, sectioning of the tissue, and preparation and the pretreatment of the sections were performed as described above.