Fig 1: The outgrowth of cholinergic neurons from the brain slices coupled with NGF microcontact prints (μCP). Brain slices (300 μm thick) of the nBM were prepared and connected to the NGF μCP, incubated for 1 week with 100 ng/ml of NGF in the medium, and then further for 2 weeks without NGF. The slices were fixed and stained for ChAT and Alexa-488 (green) and the ChAT processes were evaluated at the borders of the slices (A). (B) The outgrowth of ChAT+ neurons along the μCP (white lines, star *). Three typical outgrowths are seen, thin axons, but also thicker processes. (C–E) A typical example of an outgrowth of ChAT+ nerve fibers [(C), green, Alexa-488], co-stained with an anti-NGF antibody [(D), red, Alexa-546]. (E) Shows the merged picture and that the ChAT+ nerve fibers grow along the NGF μCP. (F–H) The selectivity of growth along the μCP. (G) An example of an incomplete printing (lanes b–d), while on a complete print, (lane a) the ChAT+ processes are seen [(F), arrows]. Quantitative analysis shows the outgrowth of 24 ± 2 ChAT+ nerve fibers per slice with a length of 286 ± 35 μm after 3 weeks in culture. Ten brain slices were prepared from five animals. Scale bar in A = 270 μm (A), 60 μm (B), 54 μm (C–E), 108 μm (F–H).
Fig 2: Re-organization within the slices placed onto the NGF μCP (A). Note some form of the re-organization of ChAT+ cholinergic neurons and fibers (B) along the μCP [(B), dotted lines, star *]. The GFAP+ cells and processes re-organized in the slice and grew on the NGF μCP [(C), dotted white line] leaving some empty areas in-between (D). For each representative two to four brain slices were prepared from two to five animals. Scale bar in A = 210 μm (A), 50 μm (B,C), 25 μm (D).
Fig 3: Co-localization of cholinergic neurons stained with ChAT (A,D,G) and with microtubule-associated protein-2 [MAP-2, (B)] or glial fibrillary acidic protein [GFAP, (E)] or the low-affinity NGF receptor p75NTR (H). Brain slices were cultured on NGF μCP for 3 weeks, fixed, and stained for ChAT (Alexa-488, green) and MAP-2, GFAP, or p75NTR (Alexa-546, red). The merged pictures (C,F,I) show the orientation of the slices, the borders (white thick dotted lines), and the suggested μCP lanes (white small dotted lines with a star *). (A) The ChAT+ processes (six lanes) grew out of the slice (arrow) along the μCP. (B) The MAP-2+ processes which fully co-localized with the ChAT+ processes and display a differential expression intensity [see as an example the white circle in (B)]. (D) ChAT+ processes (4 lanes) growing out of the slices (arrow) along the μCP. (E) shows intense GFAP+ staining within the brain slices, more extensive at the borders representing reactive astrogliosis, but nearly no immunopositive processes outside the slices [see the white circle in (E)]. (G) The immunostaining of two selected ChAT+ processes along a μCP, which fully co-localizes with the p75NTR neurotrophin receptor (H). The small arrow in (G–I) point to strong differential p75NTR immunoreactive varicosities (probably synaptic processes) within the ChAT+ processes, but also strong neuronal immunoreactive areas [white circle in (H,I)] are found. Note that all these stainings do not come from the same slice, but from four to six brain slices prepared from two to five animals. Scale bar in A = 180 μm (A–F), 36 μm (G–I).
Fig 4: Coupling of brain slices to microcontact prints. Chopper brain slices (300 μm) were prepared from the basal nucleus of Meynert (nBM) and cultured on semipermeable membrane inserts (A). The slices were coupled with the μCP of NGF (B). After 2 weeks in culture, the cholinergic neurons were stained for choline acetyltransferase (ChAT) and many cholinergic neurons survived when incubated with 100 ng/ml NGF in the medium (C), while no cholinergic neurons were visible when incubated without NGF (D). The brain slices were stained for nuclear 4′,6-diamidino-2-phenylindole (DAPI) [(E), blue] and were connected to the NGF μCP (stained by anti-NGF Alexa-546, red), showing that the slice grows directly in connection with the μCP. Scale bar in A = 500 μm (A), 130 μm (C,D), 250 μm (E–G).
Fig 5: Characterization of nerve growth factor (NGF) μCP on semipermeable membranes. Printing of collagen alone (A) or NGF alone (B) does not show any positive staining, however, NGF loaded into collagen hydrogel solution shows a typical printing when stained with Alexa-546 anti-NGF antibodies (C). The staining is specific, as it is not seen in the green channel (D). A concentration experiment shows that printing with 1,000 ng NGF per stamp (E) gives the best results, while 100 ng (F) and 10 ng (G) NGF do not show good printing. The stability of the prints (1,000 ng NGF loaded into collagen hydrogel solution) in the medium is shown in (H–J), with strong bands after printing [day 0, (H)], no change after 9 days in the medium (I), and a significant decrease after 14 days in the medium (J). The values in (E–J) give the optical density of the printed pattern from 0 (white) to 255 (black) corrected for the background and measured by computer-assisted imaging. The values are presented as mean ± SEM, with the number of independent experiments (n) and statistical differences (*p < 0.05; ***p < 0.001) with ANOVA and Fisher least significant difference (LSD) post hoc test. Scale bar in A = 175 μm (A–D), 102 μm (E–G), 175 μm (H–J).
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