Tcs sp5 aobs spectral confocal microscope

To determine target protein localization and expression patterns in hair follicles, histological tissues sections were used. At least one FU in each experimental group was fixed in 3.7% aqueous formaldehyde and embedded in paraffin using standard procedures. Typically, 18–22, 8 µm thick, longitudinal tissue sections were obtained for each FU. As a rule, the most 6-4 central sections, encompassing as much as possible the full length of, at least, the suprabulbar fiber and hair bulb regions, were used for comparative analysis. Antigen retrieval was performed using 10 mM citrate buffer in hydrated sections following standard procedures. Primary antibodies, including anti-cytokeratin 15 (CK15, clon EPR16Y, Abcam Cambridge, UK), and anti-KI67 (clon SP6, Abcam), and anti-cyclin D1 (CCDN1, clon EPR2241, Abcam) were incubated overnight at 4 °C in a wet chamber, extensively washed with PBS, incubated for 1 h at room temperature with appropriate secondary fluorescence- or HRP-labelled antibodies, and mounted in DAPI (100 ng/mL)-containing Vectashield. Confocal images were obtained using a Leica TCS SP5 AOBS spectral confocal microscope (Wetzlar, Germany) and processed using the FIJI software. Bright field images were obtained using a Nikon Eclipse Ci LED-fluorescence microscope (Tokio, Japan) coupled to a Jenoptik PROGRES GRYPHAX^® SUBRA Super HD camera.

To determine target protein localization and expression patterns in hair follicles, histological tissues sections were used. At least one FU in each experimental group was fixed in 3.7% aqueous formaldehyde and embedded in paraffin as described above. Antigen retrieval was performed using 10 mM Citrate Buffer in hydrated sections following standard procedures. Primary antibodies, including anti-Cytokeratin 15 (CK15, clon EPR16Y, Abcam) and anti-KI67 (clon SP6, Abcam), anti-E-Cadherin (ECCD1; clon 24E10, Cell Signalling), anti-TCF4 (clon D4, Santa Cruz Biotech) and anti-DKK1 (clon B7, Santa Cruz Biotech), were incubated over night at 4 °C in a wet chamber, extensively washed with PBS, incubated for 1 hour at room temperature with appropriate secondary fluorescence-labelled antibodies and mounted in DAPI (100 ng/ml)-containing Vectashield. Confocal images were obtained in Leica TCS SP5 AOBS spectral confocal microscope and processed using the FIJI software.

Aniline blue staining of plasmodesmata-associated callose of Physcomitrella leaves were performed according to the protocol described by Zavaliev and Epel (2015) , using 0.01% (w/v) aniline blue in 0.01 M K₃PO₄, pH 12. The leaves were then whole-mounted in the staining solution itself and imaged with a Leica TCS SP5 AOBS Spectral Confocal microscope (Leica Microsystems) equipped with a 20×/0.7 NA Plan-Apo dry objective lens, DIC optics, and Leica confocal software. Plasmodesmata-associated aniline blue staining was imaged (512 pixels × 512 pixels) at an excitation wavelength of 405 nm, and the emission signal was detected between 475 and 525 nm for aniline blue, and between 643 and 730 nm for chlorophyll autofluorescence. Negative controls were performed by excluding aniline blue.