Following euthanasia, ocular tissues excised from the mice of defined ages from E12.5 to adult were fixed in 4% buffered paraformaldehyde (PFA) for 1 hour at room temperature for subsequent investigation. The ocular tissues of E12.5 mice were embedded in paraffin. The paraffin sections of E12.5 mice were stained with hematoxylin and eosin. For Toluidine Blue O staining, 0.4% (w/v) Toluidine Blue O (Cas No. 92-31-9; Sigma-Aldrich, USA) was dissolved in 60% ethyl alcohol acidified with hydrochloric acid (pH 2.0). Corneas were collected with intact limbus and fixed in 95% ethyl alcohol for 30 minutes. Next, they were washed in distilled water several times and stained with 0.4% Toluidine Blue O for 10 minutes54 (link). For Alcian Blue/Safranin O staining, 1% (w/v) Alcian Blue 8GX (Cas No. 33864-99-2; Sigma-Aldrich, USA) was dissolved in 0.7 M hydrochloric acid, and 0.5% (w/v) Safranin O (Cas No. 477-73-6; Sigma-Aldrich, USA) was dissolved in 0.125 M hydrochloric acid. Corneas with complete limbus were stained in 1% Alcian Blue for 45 minutes and, after rinsing with distilled water, stained with 0.5% Safranin O for 10 minutes54 (link).The stained corneas were washed several times with distilled water, cut radially, and flattened with a coverslip. All of the samples were analyzed with a DeltaVision microscopy imaging system (Applied Precision, Issaquah, WA, USA).
Deltavision microscopy imaging system
Manufactured by Cytiva
Sourced in United States
The DeltaVision microscopy imaging system is a high-performance microscope designed for advanced fluorescence imaging applications. It provides reliable and precise image acquisition and analysis capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Safranin o, by Merck Group (1 mentions)
Alcian blue 8gx, by Merck Group (1 mentions)
Toluidine blue o, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Alexa fluor 594 conjugated goat anti mouse immunoglobulin g, by Thermo Fisher Scientific (1 mentions)
Fluoview fv1000 confocal laser scanning microscope, by Olympus (1 mentions)
Fluorescein isothiocyanateeconjugated anti mouse ccr3 antibody, by BioLegend (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
6 protocols using deltavision microscopy imaging system
1
Ocular Tissue Histological Staining
2
Cell Imaging in Culture Plates
3
Indirect Immunofluorescence Microscopy Protocol
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Cells were seeded on a confocal dish rinsed in PBS, fixed in methanol for 10 min, and then permeabilized using 0.1% Triton X-100 in 1xPBS with 30 min. The cells were blocked with 5% BSA (Sigma) for 1 h and then incubated overnight at 4 °C with the primary antibody. After washing twice in PBS, cells were incubated for 1 h with a cocktail of secondary antibodies including Donkey Anti-Mouse conjugated to tetramethylrhodamine isothiocyanate (T5393, TRITC, 1:100; Sigma) and Donkey Anti-Rabbit conjugated to FITC (F0382, 1:100; Sigma) diluted in blocking solution. Appropriate washing in PBS was performed between each step, and incubation was performed in a dark, moist chamber at room temperature. The sections were mounted with VECTASHIELD (Vector Laboratories). Images were obtained on DeltaVision Microscopy Imaging System (Applied Precision, Issaquah, WA, USA).
4
Immunostaining of H3K9me3 in Cells
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Immunostaining was performed as previously described (Maeshima et al., 2010 (link); Hihara et al., 2012 (link)). Cells were fixed in 2% formaldehyde (Wako) and permeabilized with Triton X-100. The primary and secondary antibodies were mouse anti-H3K9me3 (a generous gift from Hiroshi Kimura) and Alexa-Fluor-594-conjugated goat anti-mouse immunoglobulin G (Invitrogen) used at dilutions of 1:500 and 1:1000, respectively. Then DAPI (500 ng/ml) was added to the cells for 5 min, followed by washing with PBS prior to DNA staining. Images were obtained using a DeltaVision microscopy imaging system (Applied Precision) or a FLUOVIEW FV1000 confocal laser scanning microscope (OLYMPUS).
5
Visualizing ER-Localized HyPer-Red Biosensor
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Cells were seeded onto a confocal dish (SPL, Life Sciences, Seoul, Korea) and transiently transfected with HyPer-Red ER constructs using Lipofectamin 3000. The cells were washed twice in PBS and then cell were stained with Hoechst 33342 for 3 min. Images were obtained on DeltaVision Microscopy Imaging System (Applied Precision, Issaquah, WA, USA).
6
Immunofluorescent Staining of Ocular Conjunctiva
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The entire ocular surface, including the cornea and the conjunctiva up to the mucocutaneous junction, was dissected, as described previously. 31 Briefly, the entire eye was dissected together with the eyelid and fixed in paraformaldehyde for 1 hour. Next, under the dissecting microscope, the tissues were clipped to remove muscle and other accessory tissues so that only the conjunctival tissues remained. The conjunctival tissues were then blocked in 2% bovine serum albumin for 15 minutes, followed by permeabilization in 0.2% Triton X-100 for 15 minutes at room temperature. Subsequently, the conjunctival tissues were incubated with phycoerythrin (PE)-conjugated anti-mouse Siglec-F (552126; BD Biosciences, San Jose, CA), Alexa Fluor 488-conjugated anti-mouse CD125 antibody (558533; BD Biosciences), or fluorescein isothiocyanateeconjugated anti-mouse CCR3 antibody (144510; BioLegend, San Diego, CA) at 4 C overnight. The stained conjunctival tissues were washed three times with PBS and placed on glass slides to flatten them. A mounting medium containing 1 mmol/L DAPI (28718-90-3; Sigma-Aldrich, St. Louis, MO) was put on the conjunctival tissues. Images of the conjunctiva were analyzed using a DeltaVision microscopy imaging system (Applied Precision, Issaquah, WA).
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