I often have difficulty in immunostaining of the structure of 3-D cultured cells, often referred to as spheroids or organoids embedded in Matrigel. What kind of methods/ protocols do you usually take for the constantly good IHC result?
We usually first concentrate fresh spheroids by letting them to settle down in a tube or cryomold, than we remove the remaining cell culture medium or buffer in which the spheroids were initially suspended to obtain pure and dense population of spheroids. We then transfer the spheroids into the cryomold (if prepared in the tube), cover with Tissue-Tek® O.C.T.™ Compound and freeze in isopentan chilled with liquid nitrogen. Frozen tissue is stored at -80°C before cryosectioning. Sections are fixed with appropriate fixative and stained with antibodies as needed. I hope, this helps.
salam alikum
There are many protocols are proven effective , tha best of these three protocols from my point of view
1- 3D bulk cultures for immunohistochemistry ( Ref .Ville Härmä )
Cells culture in Millicell hanging cell culture inserts with 1.0 µm PET transparent membranes Millipore) on 6-well plates (Costar). Membranes were pre-coated with (Matrigel/medium (1:1)and incubated at 37˚C for 1 h, to prevent attachment to the membrane. Cell suspension wasmixed 1:4 with Matrigel, transferred to the coated well, and polymerized overnight at 37˚C. Cells fed every 2-3 days with fresh medium from underneath. The gels, including themulticellular structures, were excised with a scalpel and transferred into 4% paraformaldehyde for fixation. Fixed cell samples were cast into liquid agar pellets, moulded in paraffin, cut into thin sections with a microtome, and transferred onto microscope slides. The sections were first processed and stained with the IHC .
2- Three-dimensional cell culture and immunohistochemistry ( Ref .Kate Lawrenson )
Tissue culture vessels twice coated with a 1.5% of poly-2-hydroxyethyl methacrylate (polyHEMA, Sigma) solution in 95% ethanol, and allowed to dry. Before use, polyHEMA coated plates were washed with sterile PBS.Cells were trypsinised and counted, and 1×105 cells plated into polyHEMA coated P100 dishes in 25 mls complete medium. To fix the 3D cultures, spheroids were collected into a 50 ml falcon tube washed twce in PBS and fixed for 30 mins in neutal-buffered formalin (VWR, West Chester,PA, USA). Fixed 3D cultures were then processed into paraffin blocks, sectioned and stained by immunohistochemistry .
3- 3D co-cultures immunohistochemistry for Tissue freezing ( Ref. F. Duttenhoefer)
After 7 days, the scaffolds were transferred into wells
containing “Tissue freezing media” (Leica Microsystems,
Nussloch, Germany) for cryosectioning. After snapfreezing
in isopentane (Sigma-Aldrich) cooled in liquid
nitrogen, the frozen scaffolds were sectioned (10 μm)
using a Microm HM 560 microtome (Thermo Scientific,
Walldorf, Germany), and collected on Superfrost Plus®
microscope slides (Gerhard Menzel, Braunschweig,
Germany). Sections were preserved at -20 °C until use.
Cryosections were fixed in 70 % methanol for 15 min
and rehydrated in PBS for 10 min. The slides were
incubated for 30 min in methanol containing 0.3 % H2
O2 to block endogenous peroxidase, then transferred to a moist
chamber for the staining processes. After blocking nonspecific
sites with a 1 h incubation in horse serum (diluted
1:20 in PBS-Tween, Vector Labs, Burlingame, CA, USA)
samples were incubated for 30 min in the primary antibody
diluted at the optimal concentration: von Willebrand Factor
(vWF)(F8-86, Signet 115-01; Covance, Princeton, NJ,
USA) was used at 1:400 and CD146 (Abcam) at 1:1000.
αSMA (Abcam), NG2 (Abcam), and PECAM-1 (R&D
Systems) were diluted to a final concentration of 0.5 μg/
mL, 3.85 μg/mL, and 1 μg/mL, respectively. The primary
antibodies were omitted and replaced by PBS-Tween for
the negative controls. After 3 washings in PBS-Tween,
the secondary antibodies were added and incubated for
30 min. Biotinylated goat anti-rabbit (dilution 1:200, Vector
Labs: BA-1000) was used to detect the vWF antibody. A
biotinylated horse anti-mouse (Vectastain ABC kit, Vector
Labs), at a dilution of 1:200, was used to detect the CD146
and PECAM antibodies. After washing in PBS-Tween,
antibodies were visualised with the Vectastain Elite ABC
kit and DAB (both Vector Labs). Slides were cover-slipped
with Prolong Gold Antifade reagent with DAPI (for
nuclear counterstain) (Invitrogen/Molecular Probes). For
digital photographic documentation an Axioplan upright
microscope with Axiovision software (Carl Zeiss) was
used.
Facility , details of these protocols and pleas visit link
i hope help you
Good Luck
http://www.jove.com/video/5517/3d-tissue-engineered-systems-for-regenerative-approaches-drug
We more or less use the same technique as mentioned by Tomo Saric. What I would recommend in addition, is to cut the opening of the tip ca. 2-5 mm, depending on the size of your spheroids. This prevents the spheroids to be disrupted while being transferred to the cryomold. I directly pipet the spheroids from the wells they are cultivated in into a cryomold that is filled with TissueTek. Be aware of placing it in the middle of the filled TissueTek, meaning it should not touch the bottom of the cryomold nor the top of the TissueTek. The remaining medium is removed directly. This is important, otherwise you may have problems when you section the spheroids. The spheroids are frozen in liquid nitrogen atmosphere and stored at -80°C. Before sectioning, you have to adopt them to -20°C for at least 1/2h and then prepare the sections at -20°C, too.
Hi, Go. I also encountered the same problem when I did immunostaining assay for the intestinal organoids in Matrigel recently. I want to know which protocol did you take at last to solve the problem. Hope for your help.
Check this paper for a detailed protocol on immunostaining of intact organoids/spheres.
https://www.ncbi.nlm.nih.gov/pubmed/30548444
Generation of Pancreatic Ductal Organoids and Whole-Mount Immunostaining of Intact Organoids
Heyhooo, if you follow Henner Farin on Pubmed or Researchgate you probably find your answers. He designed great protocolls for that topic!
cheers
Heike