Immunofluorescence Staining of Gel-Embedded Patient-Derived Diseased Fibrovascular Tissue

Abstract

Source: Gucciardo, E., et. al., An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy. J. Vis. Exp. (2019)

This video details an immunostaining protocol for ex vivo cultures of fibrovascular tissue embedded in fibrin gel. This enables detailed visualization of vascular changes associated with proliferative diabetic retinopathy

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Whole-Mount Immunofluorescence Staining

NOTE: This protocol lasts 5 days and can be interrupted with overnight incubations where indicated. Do not let the fibrin gels dry at any point. All steps are performed at RT unless otherwise indicated.

1. Prepare the following solutions before starting the staining protocol.
   1. Post-fixation solution: prepare 50:50 acetone:methanol solution by mixing equal amounts of acetone and methanol (e.g., 50 mL). Store at -20 °C. Prepare this solution latest on the day before the staining. This solution can be stored back at -20 °C and be used for subsequent stainings.
      NOTE: Prepare this solution in fume hood as acetone and methanol are flammable and hazardous to health.
   2. Blocking solution: prepare a 15% fetal bovine serum (FBS), 0.3% octyl phenol ethoxylate solution in PBS by first adding 15% FBS to PBS. Add octyl phenol ethoxylate and stir to dissolve. Store at 4 °C.
      NOTE: This solution can be prepared in large amounts in advance, stored in 15 mL aliquots at -20 °C, and thawed before use on the day the staining protocol is started. Use freshly prepared or freshly thawed aliquots for each staining protocol.
   3. Washing solution: Prepare 1 L of PBS supplemented with 0.45% octyl phenol ethoxylate by adding 4.5 mL of octyl phenol ethoxylate to 995.5 mL of PBS. Stir to dissolve. Store at RT.
2. Lift the droplets carefully from the plate by using a stainless steel square-edged spatula. Detach the droplet first from the edges before lifting the center, and transfer to a well of 12-well plate containing 1 mL of PBS.
   NOTE: Perform post-fixation, washes and blocking steps in this plate format.
3. Post-fix the droplets with 1 mL of ice-cold 50:50 acetone:methanol solution for ~1 min (the border of the droplets will turn white).
   NOTE: Perform this step in fume hood as acetone and methanol are hazardous to health.
4. Rinse with 3 - 5 washes in 2 mL of PBS (the droplets will sink in the PBS solution when rehydration is complete).
   NOTE: Avoid touching the droplets with the pipette tip as, after post-fixation, they are sticky to plastic. Throughout the protocol, avoid aspirating post-fix, antibody, blocking and washing solutions by suction, as this may damage or completely destroy the droplets. Instead, tilt the plate and carefully remove solutions using a 500-5,000 μL pipette.
5. Centrifuge the blocking solution for 15 min at 21,000 x g and 4 °C in order to remove debris.
   NOTE: The solution can be aliquoted in 1.5 mL tubes for centrifugation. The unused solution can be stored at 4 °C and used for all relevant subsequent steps.
6. Incubate the droplets in 500 μL blocking solution for 2 h at RT.
   NOTE: To reduce the volume of blocking solution used, the plate can be tilted on a support and as little as 300 μL of solution/droplet can be used.
7. Prepare the primary antibody mixture (at least 30 μL/droplet) at appropriate dilution in blocking solution and centrifuge for 15 min at 21,000 x g and 4 °C.
8. Transfer the droplets into round (U)-bottom 96-well plate by using a round-edged spatula, and incubate with at least 30 μL/droplet of the primary antibody mixture overnight at 4 °C.
9. On the following day, transfer the droplets into a 12-well plate containing 2 mL of washing solution/well, rinse with three 5 min washes first and then with nine 30 min washes. Leave the last wash (2 mL) overnight at 4 °C.
10. On the following day, rinse three times with PBS and transfer the droplets into a round (U)-bottom 96-well plate.
11. During the washes, prepare the appropriate fluorophore-conjugated secondary antibody mixture (diluted 1:500, at least 30 μL/droplet) in blocking solution and centrifuge for 15 min at 21,000 x g and 4 °C.
12. Transfer the droplets into a round (U)-bottom 96-well plate by using a round-edged spatula and incubate with at least 30 μL of the secondary antibody mixture, for 4 h at RT, protected from light.
    NOTE: Perform all subsequent incubations and washing steps protected from light.
13. Transfer the droplets into a 12-well plate containing 2 mL of washing solution/well using a round-edged spatula, rinse with three 5 min washes first and then with four 30 min washes. Leave the last wash (2 mL) overnight at 4 °C.
14. On the following day, rinse the gels with five 30 min washes and then three times with PBS. Leave the last wash (2 mL) overnight at 4 °C.
15. On the following day, transfer the droplets into a round (U)-bottom 96-well plate by using a round-edged spatula and counterstain nuclei by incubating with 10 μg/mL Hoechst nuclear stain (at least 30 μL/droplet) for 30 minutes at RT.
    NOTE: Mounting medium supplemented with 4',6-diamidino-2-phenylindole (DAPI) for nuclei counterstaining can alternatively be used. In which case, this step and step 1.16 are skipped, proceeding directly to step 1.17.
16. Transfer the droplets into a 12-well plate containing 2 mL of PBS/well using a round-edged spatula and rinse three times with PBS.
17. Mount the droplets onto microscope slide as follows:
    1. Apply a narrow layer of quick-hardening mounting medium on the edges of a square-shaped 22 mm x 22 mm coverglass and let dry for ~1 minute. Perform this step for each drop individually, to avoid excessive hardening of the mounting medium.
       NOTE: Perform this step in fume hood as the quick-hardening mounting medium is hazardous to health.
    2. Rinse the droplet by dipping in a well of a 12-well plate containing 2 mL of deionized water and transfer onto a microscope slide, using a round-edged spatula. Remove excess water by using a small piece of absorbent paper.
    3. Dispense 15 μL of non-hardening antifade mounting medium onto the droplet.
       NOTE: Alternatively, if Hoechst nuclear stain has not been used, mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) can be used.
    4. Gently position the cover glass, with the quick-hardening mounting medium facing the microscopic slide, over the droplet and let settle.
       NOTE: The quick-hardening medium will stick to the microscopic slide and keep the droplet in place.
18. Repeat step 1.17 for all droplets. Mount two droplets on each microscope slide.
19. Let the slides air-dry at RT for ~2 h and store at 4 °C overnight. Ensure that the slides are positioned horizontally and protected from light.
20. Image the stained native or end-point FT ex vivo cultures using a confocal microscope or an upright epifluorescence microscope equipped with an optical sectioning function and 20x or 40x objective. Use tile function to capture a greater area of the tissue at once.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Material
Cell culture plates, 96-well, U-bottom	Greiner Bio-One	392-0019	Used for whole-mount immunofluorescence
Round/Flat Spatulas, Stainless Steel	VWR	82027-528	Used for whole-mount immunofluorescence
Coverslips 22x22mm #1	Menzel/Fisher	15727582	Used for mounting
Microscope slides	Fisher	Kindler K102	Used for mounting
Absorbent paper	VWR	115-0202	Used for mounting
PBS tablets	Medicago	09-9400-100	Used for preparing 1x PBS
Acetone	Sigma-Aldrich	32201-2.5L-M	Used to prepare the post-fixation solution. HARMFUL: wear protective gloves and/or clothing. Use in fume hood.
Methanol	Sigma-Aldrich	32213	Used to prepare the post-fixation solution. TOXIC: wear protective gloves and/or clothing. Use in fume hood.
Triton X-100 (octyl phenol ethoxylate)	Sigma-Aldrich	T9284	Used for whole-mount immunofluorescence. HARMFUL: wear protective gloves and/or clothing.
Hoechst 33342, 20mM	Life Technologies	62249	For nuclei counterstaining. HARMFUL: wear protective gloves and/or clothing, and eye and/or face protection.
VECTASHIELD Antifade Mounting Medium	Vector Laboratories	H-1000	Wear protective gloves and/or clothing, and eye protection. Use in fume hood.
VECTASHIELD Antifade Mounting Medium with DAPI	Vector Laboratories	H-1200	Mounting medium with nuclei counterstaining. Wear protective gloves and/or clothing, and eye protection. Use in fume hood.
Eukitt Quick-hardening mounting medium	Sigma-Aldrich	03989-100ml	TOXIC: Wear protective gloves and/or clothing, and eye protection. Use in fume hood.
Recombinant human VEGFA	R&D Systems	293-VE-010	50 ng/ mL final concentration
Recombinant human VEGFC	R&D Systems	752-VC-025	200 ng/ mL final concentration
Recombinant human TGFβ	Millipore	GF346	1 ng/ mL final concentration
Recombinant human bFGF	Millipore	01-106	50 ng/ mL final concentration
CD31 (JC70A)	Dako	M0823	Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab
CD34 (QBEND10)	Dako	M716501-2	Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab
CD45 (2B11+PD7/26)	Dako	M070129-2	Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab
CD68	ImmunoWay	RLM3161	Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab
Cleaved caspase-3 (5A1E)	Cell Signalling	9664	Used at 1:200 dilution, Goat anti Rabbit Alexa 594 Secondary Ab
ERG (EP111)	Dako	M731429-2	Used at 1:100 dilution, Goat anti Rabbit Alexa 594 Secondary Ab
GFAP	Dako	Z0334	Used at 1:100 dilution, Goat anti Rabbit Alexa 594 Secondary Ab
Ki67	Leica Microsystems	NCL-Ki67p	Used at 1:1500 dilution, Goat anti Rabbit Alexa 594 Secondary Ab
Lyve1	R&D Systems	AF2089	Used at 1:100 dilution, Donkey anti Goat Alexa 568 Secondary Ab
NG2	Millipore	AB5320	Used at 1:100 dilution, Goat anti Rabbit Alexa 594 Secondary Ab
Prox1	ReliaTech	102-PA32	Used at 1:200 dilution, Goat anti Rabbit Alexa 568 Secondary Ab
Prox1	R&D Systems	AF2727	Used at 1:40 dilution, Chicken anti Goat Alexa 594 Secondary Ab
VEGFR3 (9D9F9)	Millipore	MAB3757	Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab
α-SMA (1A4)	Sigma	C6198	Used at 1:400 dilution, Cy3 conjugated
Alexa Fluor488 Donkey Anti-Mouse IgG	Life Technologies	A-21202	Used at 1:500 dilution
Alexa Fluor594 Goat Anti-Rabbit IgG	Invitrogen	A-11012	Used at 1:500 dilution
Alexa Fluor568 Donkey anti-Goat IgG	Thermo Scientific	A-11057	Used at 1:500 dilution
Alexa Fluor568 Goat anti-Rabbit IgG	Thermo Scientific	A-11036	Used at 1:500 dilution
Alexa Fluor594 Chicken Anti-Goat IgG	Molecular Probes	A-21468	Used at 1:500 dilution
Name	Company	Catalog Number	Comments
Microscopes
Axiovert 200 inverted epifluorescence microscope	Zeiss		For imaging of the fresh and fibrin-embedded FT
AxioImager.Z1 upright epifluorescence microscope with Apotome	Zeiss		For imaging of whole-mount immunostained FT