Hormone immunolabeling in resin-embedded Arabidopsis tissues

Protocol Hormone immunolabeling in resin-embedded Arabidopsis tissues Here, we present a protocol for immunolabeling of molecules in Arabidopsis tissues. We describe steps for tissue fixation and embedding in resin of microtome-derived sections, immunolabeling using fluorescent and non-fluorescent secondary antibodies, and visualization of cytokinin and auxin molecules. This protocol is suitable to study reproductive structures such as inflorescences, flowers, fruits, and tissue-culture-derived samples. This protocol is useful for studying the distribution of a wide range of molecules including hormones and cell wall components. Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Humberto HerreraUbaldo, He´ctorRogelio Na´jeraGonza´lez, Valentı ´n Luna-Garcı ´a, Nayelli Marsch-Martı ´nez, Stefan de Folter stefan.defolter@ cinvestav.mx Highlights Plant tissue fixation and embedding in resintomakethin tissue sections Immunolabeling to detect hormones in resin sections Immunolabeling using fluorescent and non-fluorescent secondary antibodies Visualization of cytokinin and auxin molecules in plant tissues Herrera-Ubaldo et al., STAR Protocols 4, 102514 September 15, 2023 ª2023 The Author(s). https://doi.org/10.1016/ j.xpro.2023.102514 ll OPEN ACCESS Protocol Hormone immunolabeling in resin-embedded Arabidopsis tissues Humberto Herrera-Ubaldo, 1,3,4,7 He ´ctor-Rogelio Na ´jera-Gonza ´lez, 1,3,5,7 Valentı ´n Luna-Garcı ´a, 1 Nayelli Marsch-Martı ´nez, 2 and Stefan de Folter 1,6,8, * 1 Unidad de Geno ´mica Avanzada (UGA-LANGEBIO), Centro de Investigacio ´n y de Estudios Avanzados del Instituto Polite ´cnico Nacional (CINVESTAV-IPN), Irapuato, Guanajuato 36824, Me ´xico 2 Departamento de Biotecnologı ´a y Bioquı ´mica, Centro de Investigacio ´n y de Estudios Avanzados del Instituto Polite ´cnico Nacional (CINVESTAV-IPN), Irapuato, Guanajuato 36824, Me ´xico 3 These authors contributed equally 4 Present address: Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UK 5 Present address: Institute of Genomics for Crop Abiotic Stress Tolerance, Department of Plant and Soil Science, Texas, Tech University, Lubbock, TX 79409, USA 6 Twitter: @defolter_lab 7 Technical contact: [email protected];[email protected] 8 Lead contact *Correspondence: [email protected] https://doi.org/10.1016/j.xpro.2023.102514 SUMMARY Here, we present a protocol for immunolabeling of molecules in Arabidopsis tissues. We describe steps for tissue fixation and embedding in resin of microtome-derived sections, immunolabeling using fluorescent and non-fluorescent secondary antibodies, and visualization of cytokinin and auxin molecules. This protocol is suitable to study reproductive structures such as inflorescences, flowers, fruits, and tissue-culture-derived samples. This protocol is useful for studying the distribution of a wide range of molecules including hormones and cell wall components. For complete details on the use and execution of this protocol, please refer to Herrera-Ubaldo et al. (2019). 1 BEFORE YOU BEGIN Here, we describe how to perform immunolabeling of molecules in plant tissues embedded in resin (Figures 1 and 2). Depending on the antibody used, it is possible to visualize hormones such as auxin and cytokinin, or cell wall components such as mannans (Figure 3). 1–3 This protocol is not limited, and can also be used for experiments that employ other antibodies or other plant tissues from Arabidopsis or other plant species. Furthermore, variations of this protocol can also be used to visualize microtubules using whole mount immunolocalization. 4,5 Important, always investigate if an additional treatment must be performed after the fixation step to be able to detect specific molecules by immunolabeling. To detect hormones, no additional treatment is needed, however, to detect cell wall components such as mannans, an additional treatment is needed (See troubleshooting Section). 2,6 Sample embedding Timing: 3 days 1. Tissue collection and fixation. STAR Protocols 4, 102514, September 15, 2023 ª2023 The Author(s). This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1 ll OPEN ACCESS a. Collect the sample in a tube containing cold fixation buffer. Note: Dependingonthesamplesizeyoucanuse2mLor50mLtubes.Agoodvolumeratiofor solution:sampleis10:1.Forsmallsamples,theworkingvolumecouldbe1mLsolutionin2mL tubes, for larger samples (i.e., Arabidopsis inflorescences), 5 mL of solution in 50 mL falcon tubes should work. Consider collecting enough samples to have technical replicates and controls. b. Place the tube with the sample in a beaker with ice in a vacuum desiccator and apply vacuum (0.6 kPa) for 30 min. Note: The samples should sink as an indicator of good penetration of fixative. See materials and equipment Section for fixation buffer. When the aim is to observe hormones, the MTSB buffer must be included in the fixation buffer. Note: If the tissue does not sink after vacuum application, try to shake the tube to remove any air bubbles. If the samples do not sink, apply vacuum for 15 min more. c. Remove the tube from the vacuum chamber and place the tube on ice and incubate for 2.5 h. Figure 1. Overview of the immunolabeling protocol ll OPEN ACCESS 2STAR Protocols 4, 102514, September 15, 2023 Protocol Figure 2. Details of how to use and handle a 24-well plate in the immunolabeling procedure (A) Examples of base molds and embedding cassettes (A1, A2). (B) Base mold, adding 200 mL Technovit polymerization solution (B1), followed by placing the samples and then cover them with more of the same solution (B2). (C) Embedding cassette placed over the base mold with the polymerized block (C1), followed by filling the cassette with the glue Technovit 3040 (yellow) (C2). (D) Trimming of the block, block can be wetted with water (D2) to easier trim the block with a knife (D3), final result of trimmed block (D4). (E) Cassette with glued block on it can be placed in the microtome. (F) Preparation of sections with a microtome. (G) Example of putting or removing a section from the 24-well plate. (H) Side view of the 24-well plate with solution. (I and J) Examples of sections floating on the solution in a well (top view). (K and L) Examples of a typical experiment using 8 wells: two for negative controls (NC1: no primary antibody: NC2: no secondary antibody) and 6 for samples (e.g., three wild-type and 3 mutant samples). In L) the color of the NBT-BCIP staining solution can be seen (when using an alkaline phosphataseconjugated secondary antibody). (M) Example of how to do washing steps with a pipette. (N) Example of observing the samples under the microscope. ll OPEN ACCESS STAR Protocols 4, 102514, September 15, 2023 3 Protocol d. Remove fixation buffer and rinse 3 times with Milli-Q water (use the same volume as fixative used). Note: Plant tissue will still be flexible. CRITICAL: Fixation buffer is toxic due to the presence of paraformaldehyde, should be prepared in the fume hood. After use, discard properly. 2. Dehydration. In these steps the water in the sample will be substituted by another solution compatible with the resin. In this protocol we use methyl acrylate resin (Technovit 7100; See materials and equipment section) and ethanol solution. a. Remove water using a pipette. b. Add a volume (use the same volume of fixative used) of 20% ethanol, incubate for 2 h at 4C. c. Remove the solution, add a volume of 40% ethanol, incubate for 2 h at 4C. d. Remove the solution, add a volume of 60% ethanol, incubate for 2 h at 4C. e. Remove the solution, add a volume of 80% ethanol, incubate for 2 h at 4C. f. Remove the solution, add a volume of 100% ethanol, incubate for 2 h at 4C. Note: The protocol can be paused at this point. Samples in 100% ethanol can be stored at 4C covered from light for 1–2 days. Optional: pause in step e) leave in 80% ethanol for 14–17 h at 4C and continue the next day. Figure 3. Immunolabeling of hormones and cell wall components (A) Mannan polysaccharide distribution in a transverse section of an Arabidopsis gynoecium. (B–D) Changes in mannan polysaccharide distribution during septum development in wild type Arabidopsis gynoecium. 2 . (F and G) Comparison of mannan polysaccharide distribution in the septum in the wild-type Col-0 and the no transmitting tract (ntt) mutant of stage 12 gynoecia in Arabidopsis. 1 . (H–J) Distribution of trans-zeatin during gynoecium development in Arabidopsis (unpublished, de Folter laboratory). (L and M) (L) Trans-zeatin (cytokinin) and (M) IAA (auxin) distribution in longitudinal section of an Arabidopsis gynoecium with visible ovules (unpublished, de Folter laboratory). (O–V) Trans-zeatin (O-Q) or IAA (S-U) immunolabeling in maize calli during in vitro plant regeneration. 3 E,K,N,R, and V are the negative controls (no primary antibody). In A-K, O-V, a fluorescent DyLight 488 conjugated secondary antibody was used; in L-N, an AP-conjugated secondary antibody was used. Scale bars: A, H-N, O-R 50 mm, B-G 20 mm, S-V 100 mm. ll OPEN ACCESS 4STAR Protocols 4, 102514, September 15, 2023 Protocol 3. Pre-infiltration. This is an intermediate step to treat the sample with the resin. a. Prepare the pre-infiltration solution, in this case, ethanol:Technovit basic solution (1:1). b. Remove the ethanol solution from the tubes and add the same volume of pre-infiltration solution. c. Incubate for 2 h at 22C–25C. 4. Infiltration. In this step the sample will be embedded with one of the components of the resin. a. Preparation of the infiltration solution in advance. i. The Technovit infiltration solution is prepared by combining 100 mL of Technovit basic solutionand1bag(1g)ofHardenerIandmixingfor3hwithamagneticstirrer. ii. After preparation, the solution must be stored at 4Candisstableupto1month. b. Remove the pre-infiltration solution using a pipette and add the same volume of infiltration solution.Incubateforatleast2hat22 C–25C. For better results incubate for 14–17 h at 4C. See Note. Note: The protocol can be paused at this point. Samples can stay in infiltration solution for several days at 4C. The solution stays liquid. The infiltration solution causes the sample to become slightly brittle/breakable. 5. Polymerization.Inthisstep,thesamplewillbeembeddedintheresintomakesolidblocks. a. Take out the sample from the tube and place it in a petri dish. i. Use fine tweezers to dissect it and select the region of interest of the sample. ii. Dependingonthesamplesize,selecttheappropriatedisposablebasemoldsize(Electron Microscopy Sciences, USA; e.g., 5 35, 10 310 or 15 315 mm; Figure 2A). Optional: unused sample can be stored in the same tube in the same infiltration solution and stored at 4C for possible future use. b. Prepare the polymerization solution. i. Mix the Technovit infiltration solution with hardener II (15:1); mix by pipetting. ii. The polymerization solution can be used up to 15 min after preparation. See Note. Note: To avoid wasting solution, only prepare the amount you will need for the following step. After 15 min the solution will solidify, thus not usable anymore. c. Add some polymerization solution to the base mold. For the small mold bases, you can use 200 mL(Figure 2B1). For this step, you only need to cover the sample, not the entire base mold. i. Place the sample in the base mold and try to position it in the desired position. The sections will be parallel to the bottom of the mold. You can use tweezers or needles to help. In the beginning the solution has not much viscosity yet and the tissue can move a bit. ii. Wait for the solution to harden, it will take around 15 min for the solution to get viscous, to check this process you can use a needle to pinch inside the mold base and check if it is getting hard. Additionally, you can use the rest of the infiltration solution to monitor the process. iii. Once the solution is hard enough to keep the sample in its place (to check this try to move the sample with the needle) proceed to the next step. d. Place an embedding cassette over the base mold with the sample and add more polymerization solution to fill the base mold (for small base molds, you can use around 3 mL) (Figure 2C1). Note: In this way, the block with the sample will now be attached to the embedding cassette, which will serve as support for the block with sample, and is needed to mount the block on the microtome (but the support needed depends on the microtome used). Wait at least one hour before moving the mold. Alternatively, you can use Technovit 3040 (is yellow) to fill the base mold to attach it to the embedding cassette, which is a yellow (Figure 2C2). ll OPEN ACCESS STAR Protocols 4, 102514, September 15, 2023 5 Protocol e. Let the samples polymerize for 14–17 h at 28C–37C. Note: Tissues must have enough space during the fixation process, we recommend a 10:1 fixative:tissue in volume. If there is too much tissue squeezed into a tube, the fixation will be less efficient; air bubbles stay trapped in between the tissue. Furthermore, tissue can also be trapped in the tube and no sinking of the tissue can be observed. CRITICAL: Handle Technovit 3040 when liquid in the fume hood to avoid inhaling toxic vapors. CRITICAL: Be sure that the base mold and embedding cassette are compatible with the microtome. KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies 1ab: Rabbit anti-trans-zeatin riboside (1:500) OlChemIm, Czech Republic Cat#004 0312 1ab: Rabbit anti-indole-3-acetic-acid (N1) (1:500) OlChemIm, Czech Republic Cat#004 1536 Optional 1ab: rabbit anti-trans-zeatin riboside (1:500) Agrisera, Sweden Cat#AS09 414 Optional 1ab: rabbit anti-indole3-acetic-acid (N) (1:500) Agrisera, Sweden Cat#AS09 421 1ab: Rat heteromannan monoclonal antibody LM21 (1:500) Kerafast, USA (previously by PlantProbes, UK) Cat#ELD019 2ab: Goat anti-rabbit IgG (H&L), DyLight 488 conjugated (1:2000) Agrisera, Sweden Cat#AS09 633 2ab: Goat anti-rat IgM mu chain, DyLight 488 conjugated (1:2000) Abcam, UK Cat#ab98368 2ab: Goat anti-rabbit IgG antibody, alkaline phosphatase conjugate (1:2000) Merck, USA Cat#AP132A Chemicals, peptides, and recombinant proteins Technovit 7100 (resin) Heraeus Kulzer, Germany (alternative: Electron Microscopy Sciences, USA) https://kulzer-technik.com (alternative: EMS Cat#14653) Technovit 3040 (glue) Heraeus Kulzer (alternative: Electron Microscopy Sciences, USA) https://kulzer-technik.com (alternative: EMS Cat#14653) NBT-BCIP solution Merck, USA Cat#72091 Bovine serum albumin, fraction V GoldBio.com, USA CAS 9048-46-8, Cat#A-420-100 Other Vacuum pump Gast Manufacturing, USA DOA-P704-AA Desiccator NA NA Microtome Leica, Germany NA Fume hood NA NA Pipettes NA NA Bright-field, fluorescence, or confocal microscope Zeiss, Germany NA Falcon tubes 15 mL, 50 mL NA NA Tissue culture 24-well plates NA NA Tweezers NA NA Plastic molds – disposable base mold, different sizes Electron Microscopy Sciences, USA Cat#70915 Tissue-Tek process/embedding cassettes without lid Electron Microscopy Sciences, USA Cat#62520-W Microscope slides and cover slips NA NA (Continued on next page) ll OPEN ACCESS 6STAR Protocols 4, 102514, September 15, 2023 Protocol Antibody alternatives: Antibodies can be obtained from different suppliers. OlChemIm, Czech Republic: https://www.olchemim.cz/ Agrisera, Sweden: https://www.agrisera.com/ Kerafast, USA: https://www.kerafast.com/ Abcam, UK: https://www.abcam.com/ Merck, USA: https://www.merckmillipore.com/ MATERIALS AND EQUIPMENT Note: Prepare in the fume hood to avoid inhaling formaldehyde vapors. Note: Prepare paraformaldehyde stock solution: Dissolve 2 g of paraformaldehyde powder in 20 mL of hot Milli-Q water for a 10% stock solution. Add 1–2 drops of 1 M KOH solution for faster dilution. Use directly or this stock solution can be stored in 5 mL aliquots at 20C and use them within 2 weeks. Prepare in the fume hood to avoid inhaling formaldehyde vapors. Note: 10% Triton X-100 means commercially available Triton X-100 diluted 10-fold. CRITICAL: Fixation buffer is toxic due to paraformaldehyde. Continued REAGENT or RESOURCE SOURCE IDENTIFIER Fluoroshield, histology mounting medium Sigma, USA Cat#F6182 Cytoseal 60 mounting medium Electron Microscopy Sciences, USA Cat#18007 1ab: primary antibody; 2ab: secondary antibody. Fixation buffer Reagent Amount Final concentration Paraformaldehyde 4.5 mL of 10% stock 3% Microtubule-stabilizing buffer 7.5 mL 23MTSB 13MTSB Glutaraldehyde 50% solution 150 mL 0.5% 10% Triton X-100 150 mL 0.1% Milli-Q water Adjust to 15 mL Microtubule-stabilizing buffer; 23MTSB 7 Reagent Amount Final concentration PIPES 15 g 100 mM EGTA 1.90 g 10 mM MgSO 4 $7H 2 O 1.22 g 10 mM KOH 2.5 g 100 mM Dissolve in 500 mL distilled water and adjust pH to 7.0 with 10 M KOH solution. This buffer can be stored at 22C–25C for six months. ll OPEN ACCESS STAR Protocols 4, 102514, September 15, 2023 7 Protocol Absolute ethanol. Technovit 7100 (Heraeus Kulzer). Technovit 7100 Combipack contains:1 3500 mL basic solution, 5 3 1 g bags of hardener 1, 1 340 mL hardener 2. (https://kulzer-technik.com/en-kt/en-kt/products/ technovit-7100.html) Alternative: available at Electron Microscopy Sciences, USA: Catalog #14653. Infiltration solution: 100 mL of Technovit 7100 basic solution and 1 g (1 bag) of Technovit 7100 hardener I. Dissolve the powder, takes at least 3 h. Note: Store in a closed bottle at 4C. Stable up to 4 weeks. Polymerization solution: Mix infiltration solution with Technovit 7100 hardener II in a 15:1 ratio. Mix for 3 min by pipetting. It starts polymerizing in 5 min. Technovit 3040 preparation: Mix powder and liquid components in a 2:1 ratio in a fume hood as follows: Put the liquid component in a disposable 50 mL falcon tube. Add half of the powder component. Cap and mix vigorously. Add remaining of the powder component. Mix for 40 s. The mix starts polymerizing after 30 s and should be poured quickly. It will solidify within 5 min. This powder is not included in the Technovit 7100 Combipack. Note: Prepare in the fume hood to avoid inhaling formaldehyde vapors. Tween 20. Blocking solution: 1% albumin fraction V from BSA in 13MTSB (for fluorescent antibodies). Blocking solution: 1% albumin fraction V from BSA in 13PBS (for non-fluorescent antibodies). Primary antibody (1ab) solution: Dilute primary antibodies in blocking solution before using. Primary antibody concentration should be determined experimentally and can vary between 1:20 and 1:1000 since it is heavily dependent on epitope concentration. Secondary antibody (2ab) solution: Secondary antibody is diluted in a range from 1:200 to 1:2000 in blocking solution right before use. If provided, follow manufacturer’s instructions. Note: Prepare only the amount needed per experiment, usually 500 mLpersample.Donot store diluted antibody solutions. Detection buffer for NBT-BCIP: 100 mM Tris-HCl pH 9.5, 100 mM NaCl, 50 mM MgCl 2 .Thisbuffer canbestoredat22 C–25C for six months. Phosphate buffered saline; 103PBS Reagent Amount Final concentration NaCl 80 g 1.3 M KCl 2 g 27 mM Na 2 HPO 4 14.4 g 100 mM KH 2 PO 4 2.4 g 18 mM Dissolve in 800 mL distilled water and adjust pH to 7.4 with 1 M HCl, adjust to 1 L, autoclave at 121C for 15 min. This buffer can be stored at 22C–25C for six months. Tris-buffered saline; 103TBS Reagent Amount Final concentration Tris 60.6 g 0.5 M NaCl 87.6 g 1.5 M Dissolve in 800 mL distilled water and adjust pH to 7.5 with 1 M HCl, adjust to 1 L. This buffer can be stored at 22C–25C for six months. ll OPEN ACCESS 8STAR Protocols 4, 102514, September 15, 2023 Protocol For some samples, it is recommended to do a two-step resin inclusion; the first step will generate only a resin block with the sample, which can then be trimmed to generate a cube withthesampleintherightangle,whichcanthenbegluedinthecorrectorientationonthe embedding cassette. Problem 5 Antibody storage. Potential solution To avoid antibody degradation due to the frequent freezing/unfreezing, we recommend preparing single-use aliquots with 5 or 10 mL of the antibody. Long time storage at 20C; short term storage at 4Cisfine. RESOURCE AVAILABILITY Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Stefan de Folter ([email protected]). Materials availability This study did not generate new unique reagents. Data and code availability This study did not generate new data or code. ACKNOWLEDGMENTS We thank Silvia Moschin from the Barbara Baldan laboratory (University of Padova, Italy) for discussions and improvements to the protocol. H.H.U. and V.L.G. were supported by the Consejo Nacional de Humanidades, Ciencias y Tecnologı ´as (CONAHCYT; Mexico) with a PhD fellowship (243380 and 487657, respectively) and H.R.N.G. with an MSc fellowship (455156). The work in the S.d.F. laboratory was financed by the CONAHCYT grants CB-2012-177739 and CB-2017-2018-A1-S-10126, and N.M.M. by the CONAHCYT grants CB-2015-255069 and CF-2023-G-219. S.d.F. is grateful for participation in the European Union projects H2020-MSCA-RISE-2020 EVOfruland (101007738) and H2020-MSCA-RISE-2019 MAD (872417). AUTHOR CONTRIBUTIONS Conceptualization, H.H.U., H.R.N.G., and S.d.F.; investigation, H.H.U., H.R.N.G., and V.L.G.; writing – original draft, H.H.U. and H.R.N.G.; writing – review and editing, H.H.U., H.R.N.G., V.L.G., N.M.M., and S.d.F.; funding acquisition, N.M.M. and S.d.F. DECLARATION OF INTERESTS The authors declare no competing interests. REFERENCES 1. Herrera-Ubaldo, H., Lozano-Sotomayor, P., Ezquer, I., Di Marzo, M., Cha ´vez Montes, R.A., Go ´mez-Felipe, A., Pablo-Villa, J., Diaz-Ramirez, D., Ballester, P., Ferra ´ndiz, C., et al. (2019). New roles of NO TRANSMITTING TRACT and SEEDSTICK during medial domain development in arabidopsis fruits. 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CSH Protoc. 2008. pdb.prot4946. https://doi.org/10. 1101/pdb.prot4946. ll OPEN ACCESS 16 STAR Protocols 4, 102514, September 15, 2023 Protocol