mouse monoclonal anti βiii tubulin  (R&D Systems)

Journal: International Journal of Molecular Sciences

Article Title: Regulatory Peptide Pro-Gly-Pro Accelerates Neuroregeneration of Primary Neuroglial Culture after Mechanical Injury in Scratch Test

doi: 10.3390/ijms252010886

Figure Lengend Snippet: Immunocytochemical staining of neuroglial culture for GFAP (green) and β-III tubulin (red). Nuclei were stained with Hoechst 33342. Fluorescent images of the cell culture were obtained at day 3 after the scratch, i.e., 7 DIV ( A , B ), and at day 7 after the scratch, i.e., 10 DIV ( C , D ). The peptide PGP was added within 3 days ( B ) or 6 days after the scratch ( D ). Addition of the peptide reduced astrogliosis and increased migration of neurons into the damaged area; many nuclei were observed at the scratch border. The diagram below shows the semiquantitative representation of this process. The ratio of GFAP ( E ) and β-III tubulin ( F ) fluorescence to Hoechst 33342 fluorescence. The ratio of β-III tubulin fluorescence to GFAP fluorescence ( G ). * p < 0.05; ** p < 0.01; *** p < 0.005. There was a decrease in total GFAP fluorescence and an increase in total β-III tubulin fluorescence after adding the peptide PGP; the β-III tubulin/GFAP ratio significantly increased on the 3rd day after the scratch, i.e., in the acute period of mechanical injury. The decrease in β-III tubulin levels on the 7th day was associated with the process of neuroregeneration and active migration of cells to the scratch area. Data were acquired using an inverted fluorescence microscope Zeiss Axiovert-200 (Carl Zeiss, Jena, Germany), with objective 40×/NA = 1.35 (oil) and MetaFluor software the version 7.7.0.0 (Molecular Devices, San Jose, CA, USA acquiring images of the entire observed area. The recorded images were processed in MetaFluor Analyst software the version 1.0.93 (Molecular Devices, San Jose, CA, USA) and have been converted into a Microsoft Excel table (version 2016) with total values of fluorescence intensity in each frame. In one confocal dish, six to eight photographs were taken (on both sides of the scratch in random places). In each group, there were three confocal dishes. Thus, at least 18 photographs were taken in each group. Data were analyzed using the ordinary two-way ANOVA with Sidak’s multiple comparisons test. The normality of distribution was assessed using the D’Agostino & Pearson test. Data are presented as the mean ± SD. The results were considered statistically significant with p < 0.05. Representative images were acquired using an LSM 880 scanning laser confocal microscope equipped with an AiryScan module and GaAsP detector (Carl Zeiss, Jena, Germany) with a Plan-Apochromat 40×/1.2 mm Corr DIC M27 multi-immersion objective. The ZEN Black tile scanning function was used to stitch four separate images in each panel. A scale bar is shown in the images. The yellow double arrow corresponds to the width of the scratch.

Article Snippet: The cells were incubated overnight at 4 °C with anti-β-III tubulin (2G10) mouse monoclonal antibodies (Thermo Fisher, #MA1-118; dilution of 1:100) and anti-GFAP chicken polyclonal antibodies (Thermo Fisher, #PA1-10004; dilution of 1:1000).

Techniques: Staining, Cell Culture, Migration, Fluorescence, Microscopy, Software