Corneal explants were fixed in 4% paraformaldehyde overnight, transferred to 30% sucrose/PBS solution overnight and embedded in OCT compound (Tissue-Tek, Sakura). Ten-micron-thick transversal frozen sections were cut with a cryo-microtome. Sections were stained with 1 μg/ml bis-benzimide Hoechst (Sigma-Aldrich) and mounted with Vectashield (DAKO). For the immunofluorescence CD34 staining, sections were thawed for 15 min at room temperature, washed in PBS for 15 min to remove OCT, permeabilized (0.1% Triton) for 15 min at room temperature and blocked in 2.5% horse serum/0.5% BSA/0.1% Triton for 30 min. Primary antibodies were incubated on sections overnight at 4 °C, and the secondary antibody incubated 90 min, at room temperature prior to Hoechst labeling and mounting. For the human corneas, the primary antibody used was 1:100 mouse anti-human CD34 (clone B1-3C5; AbCam, Cambridge, UK) and the secondary antibody 1:500 goat anti-mouse IgG-Alexa546 (Molecular probes, Invitrogen). For the dog corneas, the primary antibody used was 1:100 mouse anti-dog CD34-PE (clone 1H6, eBioscience) and the secondary antibody 1:500 goat anti-mouse IgG-Alexa Fluor 546 (Molecular probes, Invitrogen). Observations and acquisitions were performed with either a Zeiss LSM510 or a LEICA SP5 laser scanning confocal microscope. Image analyses were performed using MetaMorph software.
Vectashield
Manufactured by Agilent Technologies
Sourced in Denmark
Vectashield is a mounting medium designed for fluorescence microscopy. It is formulated to preserve fluorescence signals and prevent photobleaching of fluorescent dyes or proteins.
Automatically generated - may contain errors
Lab products found in correlation
Bisbenzimide hoechst, by Merck Group (1 mentions)
Lsm 510, by Leica camera (1 mentions)
Goat anti mouse igg alexa546, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Phalloidin tetramethyl rhodamine, by Merck Group (1 mentions)
Apotome system, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Fluorescence microscope, by Leica Microsystems (1 mentions)
Axio imager z2, by Zeiss (1 mentions)
6 protocols using vectashield
1
Corneal Immunofluorescence Staining Protocol
2
Immunostaining Protocols for Drosophila Testes and Antennae
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Testes from young adult flies or pupae were dissected in PBS, opened with a tungsten needle on a slide, and squashed. Slide was frozen in liquid nitrogen and coverslip removed. Samples were fixed 10 min in methanol 100% at −20°C, washed in PBS and blocked for 1 h in PBS/0.1% BSA. Primary antibodies were incubated in blocking solution overnight at 4°C or 1 h at RT (4 h for acetylated tubulin). For acetylated tubulin, testes were kept in PBS for 1 h at 4°C to depolymerize cytoplasmic microtubules before fixation.
For both fixations, fixed testes were washed in PBS and incubated for 1 h in secondary antibodies diluted in PBS. Slides were washed in PBS, incubated in Hoechst 1/1,000 for 15 min at RT, washed in PBS, and rinsed in ultrapure water. Slides were mounted using Dako or Vectashield.
Antennae were processed as previously described (Vieillard et al., 2015 (link)). In brief, Drosophila heads from 38- to 45-h pupae were dissected in PBS, fixed for 1 h in PBS/4% PFA, and washed in PBS. Antennae were blocked for 1 h in PBS/0.3% Triton X-100/3% BSA/5% NGS and incubated in primary antibodies diluted in blocking solution for 48 h at 4°C. Samples were washed three times in PBS and incubated in secondary antibodies diluted in PBS for 48 h at 4°C. Antennae were washed three times in PBS and mounted in Vectashield.
For both fixations, fixed testes were washed in PBS and incubated for 1 h in secondary antibodies diluted in PBS. Slides were washed in PBS, incubated in Hoechst 1/1,000 for 15 min at RT, washed in PBS, and rinsed in ultrapure water. Slides were mounted using Dako or Vectashield.
Antennae were processed as previously described (Vieillard et al., 2015 (link)). In brief, Drosophila heads from 38- to 45-h pupae were dissected in PBS, fixed for 1 h in PBS/4% PFA, and washed in PBS. Antennae were blocked for 1 h in PBS/0.3% Triton X-100/3% BSA/5% NGS and incubated in primary antibodies diluted in blocking solution for 48 h at 4°C. Samples were washed three times in PBS and incubated in secondary antibodies diluted in PBS for 48 h at 4°C. Antennae were washed three times in PBS and mounted in Vectashield.
3
Fluorescent Immunolabeling of Primary AM
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Primary AM treated or not with FITC-SiO2 NPs grown onto coverslips or cytospined cells from BAL were fixed with formalin (10 min room temperature). After blocking (1% BSA-PBS), cells were incubated with rat IgG anti-mouse CD45 antibody (BD Biosciences) followed by Alexa-664-Fluor coupled secondary antibody (Invitrogen) (membrane staining). Phalloidin–tetramethyl-rhodamine (Sigma) was used for labelling cytoplasm. Nuclei were stained with DAPI (Invitrogen). The preparations were mounted with Vectashield (Dako) mounting media and were analysed under structured illumination (ApoTome system, Zeiss) using the corresponding filter for each fluorochrome. Images were recovered and analysed by the AxioVision programme and control cells (not treated with FITC-SiO2 NPs) were included to measure fluorescence background. Percentages of positive cells for each preparation were obtained after counting of at least 100 cells in several fields. At least two independent experiments were performed. Representative images are shown in Figures 3 and 7 .
4
Intracellular Delivery and Permeability of TAT-TVTSP
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Cells were plated onto coverslips in 24-well plates, and TAT-TVTSP-FAM (Supplementary Figure 2 ) was added for the indicated times. After removal of the medium, the cells were fixed with 4% PFA (Sigma-Aldrich, St. Louis, MO, USA) for 15 min. The medium was replaced by 0.1% BSA in PBS, and the cells were mounted on slides with Vectashield (DAKO, Glostrup, Denmark). FAM signals were photographed using a fluorescence microscope (Leica Microsystems, Wetzlar, Germany). The tissue permeability of TAT-TVTSP was also examined in a mouse model. Five-week-old female mice habituated for 1 week were intranasally administered TAT-TVTSP-FAM or saline. The tracheas and bronchioles were excised 2 h later and were immediately frozen in optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA, USA) and sectioned at a thickness of 10 µm. The sections were fixed with 4% PFA for 15 min, washed with 0.1% BSA in PBS, and mounted with Vectashield. Intracellular fluorescence signals were observed using a fluorescence microscope.
5
Immunostaining of Hydrocephalic Mouse Brains
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Mice (hydrocephalic sham-injected mice, n = 11; hydrocephalic hyh mice transplanted with BM-MSC, n = 12; non-hydrocephalic mice, n = 5) were sacrificed under anesthesia with Dolethal (sodium pentobarbital; Vétoquinol, Lure, France; intraperitoneal administration, 0.2 mg/g body weight) and transcardially perfused with 4% buffered paraformaldehyde. Fixed brains were removed and post-fixed in the same solution for 24 h at 4 °C. The brains were sectioned with a vibratome (80-μm-thick sections) or were cryoprotected in 30% sucrose to obtain frozen sections (60-μm-thick). Type of sections and thickness were selected accordingly to obtain the best immunolabeling depending on the used antibodies and to maintain the section integrity. Sections of brains and slides containing isolated BM-MSC were immunostained with specific primary antibodies and appropriate fluorescent secondary antibodies (Table 1 ), using DAPI as nuclear staining, and mounted in Vectashield (Dako, Glostrup, Denmark).
6
Differentiation of Neural Stem Cells
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After 7 days in culture, PCL fiber mats and seeded NSPC were isolated from the culture medium. The cells present in this culture were fixed during 15 min in a 2% paraformaldehyde solution (prepared in phosphate buffer (PBS) at pH 7.4), and then postfixed during 15 min using a 4% paraformaldehyde solution (prepared in PBS at pH 7.4). Fixed cells were processed for double immunofluorescence analysis adding both anti-βIII tubulin and anti-GFAP antibodies, and DAPI marker. The dilution of antibodies was 1:750, using a buffer solution 0.1 M Tris buffer at pH 7.8, prepared 0.7% of non-gelling seaweed gelatin and lambda carrageenan, and 0.5% of Triton X-100. The secondary antibodies, conjugated with Alexa Fluor 488 or 594, in a 1:500 dilution, were used. Incubation of antibodies were carried out during 18 h at room temperature. Fixed cells in the mats were coverslipped by using a mounting medium (Vectashield, Dako, Santa Clara, CA, USA) and inspected under an epifluorescence microscope (AxioImager Z2, Zeiss, Aalen, Germany) in order to study differentiated NSPC by using the multidimensional acquisition software AxioVision Rel version 4.6 (Zeiss, Aalen, Germany).
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