I have cryosectioned a spheroid which was grown in collagen and agarose and then embedded in OCT Tissue Tek solution before freezing in a dry ice ETOH bath of liquid N2. The sections are very clean, easy to cut (20um thick) and I can visually see the spheroid on the SuperFrost Plus slide.
However when I perform immunofluorescence or H&E staining, I feel like I am washing away the complete section. I know that the Thermo Fisher SuperFrost slides which I use are designed for adhering to tissue - however is the spheroid too small/ too different to tissue that is is not sticking to the slide? (My spheroids are 200um in diameter).
Wondering if anyone has experienced something similar when staining sectioned spheroids. Thank you for any help in advance!
slides must be superfrost PLUS (positively charged) to cause tight adhesion of tissue sections. Spheroid/organoid sections adhere just fine. check out our papers, we used the same technique PMID: 26283078, PMID: 31210100. Your slides are probably just Superfrost (this does not mean positively charged). change the slides and repeat, it will work. Don't worry about the small size of sections, they stick to + slides very well. The problem is caused by wrong slides, i believe. Hope it helps, good luck
slides must be superfrost PLUS (positively charged) to cause tight adhesion of tissue sections. Spheroid/organoid sections adhere just fine. check out our papers, we used the same technique PMID: 26283078, PMID: 31210100. Your slides are probably just Superfrost (this does not mean positively charged). change the slides and repeat, it will work. Don't worry about the small size of sections, they stick to + slides very well. The problem is caused by wrong slides, i believe. Hope it helps, good luck
Hello,
Please you look at this articles
Article Into the depths: Techniques for in vitro three-dimensional m...
3-D culture methods
Hydrogels containing 2% agarose were created from micromolds (No. 24–9; Microtissues, Inc., Providence, RI). Hydrogels were equilibrated with appropriate cell culture medium for a minimum of 30 min prior to cell seeding (16) into hydrogels at various densities (Table 1). After seeding, cells settled to the bottom of the small recesses and self-assembled into spheroids over the course of 24 h.
Microtissue histology
Microtissues within agarose hydrogels were fixed in 10% neutral buffered formalin (No. 225; Fisher Scientific, Agawam, MA) for a minimum of 24 h prior to dehydration and processing. Microtissues within the agarose molds were then embedded in Technovit 7100 glycol methacrylate (No. 14653l; Electron Microscopy Sciences, Hatfield, PA) per manufacturer’s specifications with one modification. Samples were partially submerged in the infiltration solution and hardener 2 mixture to allow the sample to firmly attach to the bottom of the histology mold prior to filling the mold completely and adding a block holder. Samples were sectioned at a thickness of 3 μm, after which sections were mounted on slides and stained with hematoxylin and eosin (H&E) for histological examination.
Preparation of frozen sections
Microtissues within agarose hydrogels were fixed in 10% neutral buffered formalin overnight at 4°C. Following fixation and 2 washes with phosphate-buffered saline (PBS), samples were placed in a 15% sucrose/PBS solution for at least 3 h at room temperature and then in a 30% sucrose/PBS solution overnight at 4°C. Samples were embedded in optimal cutting temperature compound (OCT) (No. 14–373–65, Tissue–Tek; Fisher Scientific), frozen on dry ice before sectioning, and stored at −80°C until use. Samples were sectioned at 8 μm and affixed to Superfrost plus slides (No. 12–55–010; Fisher Scientific) before storage at −80°C until immunostaining.
Immunofluorescence of frozen sections
Frozen sections affixed to slides were allowed to warm to room temperature and then fixed for 5 min in 4% paraformaldehyde or 10% neutral buffered formalin Slides were washed with PBS 3 times at room temperature and then permeabilized with 0.25% Triton X-100 for 10 min at room temperature. Samples were washed in PBS as before and then blocked with 1% goat serum, 3% BSA, and 0. M glycine for 1 h at room temperature Slides were incubated overnight at 4°C with same anti-E-cadherin antibody (1:500 dilution) used for immunohistochemistry as described above. Following incubation, slides were washed in PBS as described above and incubated with anti-mouse secondary antibody (1:1000 dilution) (No A21236; Life Technologies) for 2 h at room temperature. For F-actin staining to visualize the cytoskeleton, sections were permeabilized as above and incubated with rhodamine phalloidin (1:500 dilution) (R415; Life Technologies) for 25 min. Slides were washed as described above and stained with Hoechst 33342 (1:1000 dilution) (No. H1399; Life Technologies) for 30 min at room temperature. Coverslips were mounted using hard set mounting media (No. H-1400; Vector Laboratories) and stored in the dark at 4°C until imaging.
Immunofluorescence of whole spheroids
Whole microtissues in agarose hydrogels were fixed in 10% neutral buffered formalin overnight at 4°C. Following fixation, gels were washed with PBS 3 times for 5 min each at room temperature with gentle shaking. Spheroids were permeabilized by incubation in 0.25% Triton X-100 in PBS for 2 h at room temperature and washed as described above. Samples were blocked for 1 h at room temperature with 1% goat serum, 3% BSA, and 0.3 M glycine, followed by incubation with the same anti-E-cadherin antibody (1:500 dilution) mentioned above overnight at 4°C. Samples were then washed in PBS as previously described and incubated with anti-mouse secondary antibody (1:1000 dilution) for 2 h at room temperature followed by washes as described above, counterstained with Hoechst 33342 (1:2000 dilution) for 2 h at room temperature followed by additional PBS washes, and finally, protected from light during storage at 4°C until clearing. For Hoechst staining alone, previously fixed microtissues stored at 4°C were stained with Hoechst 33342 (1:2000 dilution) in PBS for 2 h at room temperature.
https://europepmc.org/article/pmc/pmc6117202
Preparation of Three-Dimensional (3D) Human Liver (HepaRG) Cultures for Histochemical and Immunohistochemical Staining and Light Microscopic Evaluation
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