Diamidino 2 phenylindole dihydrochloride dapi

Samples of ovaries were fixed in 4% formaldehyde. After fixation, they were rinsed, dehydrated in a graded series of ethanol, infiltrated, and embedded in histocryl acrylic resin (Agar Scientific). Semithin serial sections were stained in the dark with fluorescence dyes: diamidino-2-phenylindole dihydrochloride (DAPI, 3:100 for 45 min; Sigma-Aldrich) and with propidium iodide (1:800 for 60 min; Sigma-Aldrich). DAPI detects deoxyribonucleic acid (DNA). propidium iodide detects ribonucleic acid (RNA) and DNA. Sections were photographed using a Leica DMR epifluorescence microscope (FLM) equipped with appropriate filters. The one-step procedure of silver impregnation described in Biliński and Bilińska (1996 (link)) to detect nucleolar organizers and Ag-NOR proteins was also applied. Images of silver stained semithin sections were enhanced with differential interference contrast (Nomarski’s contrast) and were taken under the same microscope.

Proximal, mid and distal segments of the SI were fixed with 4% paraformaldehyde (PFA) from 3 h to overnight at 4 °C, embedded in OCT and kept at −80 °C until sectioning. Sections (7 µm thick) were prepared with a cryotome (Cryostat CM 3050S).
Images from Hematoxyline QS (Vector) stained were acquired using NDP Zoomer Digital Pathology (Hamamatsu) and subsequently analysed in NDP.view2 software.
To perform immunofluorescence, sections were blocked and permeabilized in 10% adult bovine serum (Sigma), 5% skim milk (Sigma) and 0.3% Triton X-100 in PBS (blocking buffer) for at least 1 h at 4 °C. Primary antibodies (listed in Supplementary Table 3) were incubated overnight in blocking buffer at 4 °C. Alexa-Fluor-Conjugated secondary antibodies (indicated in Supplementary Table 3) were incubated for 1–2 h at room temperature in 10% adult bovine serum and 0.1% bovine serum albumin (BSA) (Sigma) in PBS. Diamidino-2-phenylindole dihydrochloride (DAPI; 1 µM; Sigma) was used to counterstain nuclei in the indicated experiments. Images were acquired using laser-scanning confocal microscopes (Leica TSC SP8 and Zeiss LSM800). All images were subsequently analysed with Fiji software.

Formation
and distribution of focal adhesions in hMSC-nanofunctionalized microparticle
spheroids were qualitatively assessed via confocal fluorescence microscopy
analysis. After 4 and 24 h in culture, the spheroids were fixed with
warm 10% FA in DPBS solution for 20 min and permeabilized using a
0.1% v/v solution of Triton-X100 (Sigma-Aldrich) in Milli-Q water
for 30 min. After this, an unspecific binding blocking step of 1 h
was performed in a casein-based blocking serum (CAS-Block Histochemical
Reagent; Thermo Fisher Scientific). The spheroids were then incubated
overnight with an Alexa Fluor 647-conjugated monoclonal antivinculin
antibody (ab196579, Abcam) solution in a 0.1% w/v bovine serum albumin
(BSA; VWR) solution in phosphate-buffered saline (PBS) with a 1:100
dilution to visualize focal adhesions. At the same time, cell nuclei
were counterstained overnight using diamidino-2-phenylindole dihydrochloride
(DAPI; Sigma-Aldrich) at a dilution of 1:200 in 0.1% BSA in PBS. The
imaging was performed using a confocal laser scanning electron microscope
(TCS SP8 STED, Leica Microsystems), acquiring z-stacks of 4 μm
thickness for each image.

Actin cytoskeleton was stained to evaluate the cell morphology, stress fibre formation, and orientation in order to exclude harmful effects of ES. Osteoblasts were washed with phosphate buffered saline (PBS, Merck KGaA, Darmstadt, Germany) and fixed in 4% paraformaldehyde for 10 min at room temperature (RT). Cells were washed in PBS and incubated with 0.5% Triton-X (Merck KGaA, Darmstadt, Germany) in PBS for five minutes at RT for permeabilisation. Afterwards, the osteoblasts were rinsed with PBS and incubated with 100 nM Acti-stain 488 fluorescent phalloidin (Cytoskeleton, Denver, CO, USA) for 30 min at RT, protected from light. The osteoblasts were washed three times with PBS, and the cell nuclei were stained with diamidino-2-phenylindole dihydrochloride (DAPI, Merck KGaA, Darmstadt, Germany) for 5 min. The images were captured with a Leica DMI 6000 (Leica Microsystems, Wetzlar, Germany) with 200× magnification.

The intracellular inclusions were stained with the green-fluorescent dye 4,4 -difluoro-1,2,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (Bodipy), which stains lipids, membranes, and other lipophilic compounds. For staining, cells were washed twice with PBS, fixed for 10 min with 4% PFA, and incubated with 0.05% Triton X (Merck KGaA, Darmstadt, Germany) for 5 min at RT to permeabilize the cell membrane. Then, the cells were washed three times with PBS and 2.5 µg/mL Bodipy solution (in 150 mM NaCl, Thermo Fisher Scientific Inc., Waltham, MA, USA) was added for 20 min. Consecutively, cells were washed twice with PBS and nuclei staining was performed with diamidino-2-phenylindole dihydrochloride (DAPI, Merck KGaA, Darmstadt, Germany) for 5 min at RT. Images were captured with a fluorescence microscope (Nikon ECLIPSE TS100, Nikon GmbH, Duesseldorf, Germany) at a 400× magnification.

After 28 days of long-term BMP-2 exposure, the cytoskeleton was stained to determine morphological changes. Cells were washed with PBS and fixed in 4% paraformaldehyde for 10 min at RT. Afterward, the cells were washed with PBS and incubated with 0.05% Triton X (Merck KGaA, Darmstadt, Germany) for 5 min at RT to permeabilize the cell membrane. After two washing steps with PBS, the pre-osteoblasts were incubated with 100 nM Acti-stain 488 phalloidin (Cytoskeleton, Denver, CO, USA) for 30 min at RT and protected from light. The cells were washed three times with PBS. The staining of the nuclei was done with diamidino-2-phenylindole dihydrochloride (DAPI, Merck KGaA, Darmstadt, Germany) for 5 min at RT. Images were captured with a fluorescence microscope (Nikon ECLIPSE TS100, Nikon GmbH, Düsseldorf, Germany) at a 400× magnification.