Examining Gene Expression Patterns Through Whole-Mount In Situ Hybridization

Chapter 19 Examining Gene Expression Patterns Through Whole-Mount In Situ Hybridization Jeffery R. Barrow Abstract RNA in situ hybridization is a practical technique that allows investigators to observe temporal and spatial gene expression at the RNA level in the context of whole embryos or tissues. One powerful application of in situ hybridization is to observe the consequences of genetic, toxicologic, or environmental perturbations on gene expression or morphogenesis during development. Herein, I will review the procedure to perform nonradioactive, in situ hybridization on whole-mount mouse or chick embryos. Key words In situ hybridization, Whole mount, Chick, Mouse, Embryo, Nonradioactive, Riboprobe, Digoxigenin 1 Introduction RNA in situ hybridization is a molecular approach to observe RNA gene expression in the context of a whole embryo or intact tissue. In situ hybridization was initially performed to detect amplified rDNA sequences in nuclei of ovarian cells with radioactive rRNA probes in the African clawed toad, Xenopus laevis [1]. Since then, in situ hybridization has been extensively used to detect mRNA expression in embryos, tissues, or tumors [2–5]. The ability to detect mRNA expression has played a critical role in understanding embryonic defects due to genetic or toxicologic manipulation. Generally speaking, RNA in situ analysis is used in two ways to examine the consequences of genetic or toxicologic challenge. First, in situ can be used to label a given embryonic anlage such as the notochord (marked by Brachyury, Fig. 1a, b; see also [6]), apical ectodermal ridge (marked by Fgf8 [7, 8]), and floor plate of the neural tube (marked by Nkx2.2 [9]). One can then examine morphogenetic differences of these marked tissues between experimental and control specimens. For example, in Fig. 1a, b, wild-type and Vangl2lp/lp-mutant embryos have been subjected to Brachyury (T) in situ hybridization. One can discern Jason M. Hansen and Louise M. Winn (eds.), Developmental Toxicology: Methods and Protocols, Methods in Molecular Biology, vol. 1965, https://doi.org/10.1007/978-1-4939-9182-2_19, © Springer Science+Business Media, LLC, part of Springer Nature 2019 281 282 Jeffery R. Barrow Fig. 1 In situ hybridization of early mouse embryos with Brachyury (T) probe. (a, b) Control embryo (a) and Vangl2 mutant embryo (homozygous for the Looptail (lp) allele of Vangl2) (b) have been subjected to in situ hybridization with the T probe. Note that in contrast to strong convergence along the mediolateral axis and extension along the anteroposterior axis of notochord cells in controls (a), the T-positive cells exhibit weak convergence and extension as evidenced by scattered cells, arrowheads (b). (c–e) Control embryo (c), Wnt5a −/−/Wnt5b −/− double mutant (d), and Wnt3a −/− mutant embryos at E9.0 and subjected to in situ hybridization with the T probe. Note strong T staining in the tail bud (arrows) of the controls and Wnt5a/Wnt5b double mutants (c, d), but complete absence in the tailbud of Wnt3a mutants (arrow, e) that T-expressing notochord cells of the Vangl2lp/lp mutants are not converging and extending properly along the ventral midline of the embryo (Fig. 1b; see also [6]). Thus, one can discern that loss of Vangl2 function affects cell behavior of notochord cells during development. Alternatively, in situ hybridization can be used to examine regulatory interactions due to genetic or toxicologic manipulations. For example, Wnt3a and Wnt5a/Wnt5b mutants both exhibit body axis truncations just distal to the rib cage ([10], JRB, unpublished observations). Comparing Brachyury (T) expression in both mutant classes, one observes strong expression in the tail bud of gastrulating Wnt5a-/Wnt5b-double mutant embryos that is comparable to wildtype embryos (arrows, Fig. 1a, b). However, tail buds of Wnt3a mutants completely lack T expression (arrow, Fig. 1c). The basis for the loss of T expression is due to it being a direct target of Wnt3a/βcatenin signaling [11], whereas Wnt5a and Wnt5b are thought to signal through β-catenin-independent pathways [12]. Thus, the two Whole-Mount In Situ Hybridization 283 mutants exhibit a similar embryonic phenotype (data not shown) but arrive there by distinct molecular pathways. In situ hybridization has also been powerful tool to determine regulatory and morphogenetic consequences of toxicologic challenge during embryogenesis. For example, exposure of embryos to various teratogens such as ethanol, retinoic acid, and mercury results in characteristic embryonic defects [13–22]. Many of the molecular or morphogenetic underpinnings have been characterized through in situ hybridization. Techniques such as immunohistochemistry can also provide important information regarding temporal and spatial expression of genes including subcellular localization of the gene products. However, it can be time-consuming and expensive to generate antibodies directed against a particular gene product. In contrast, developing an RNA probe for in situ hybridization is very rapid and relatively inexpensive. Herein, I will be reviewing the process of performing in situ hybridization on whole-mount mouse or chick embryos. I will briefly discuss the generation of nonradioactive antisense RNA probes containing the hapten, digoxigenin (DIG), although other hapten-modified nucleotide-based (fluorescein, dinitrophenol, biotin) riboprobes have been used with success [23]. I will discuss procedures used to hybridize the probe to endogenous mRNA species in embryos and detection with alkaline-phosphatase conjugated anti-DIG antibodies. 2 Materials 2.1 Embryo Collection Reagents required for embryo collection: 2.1.1 Phosphate Buffered Saline (PBS) In this protocol, one uses a large volume of PBS or PBS-based solutions. It is therefore prudent to make a 10× PBS stock. Take large beaker with ~700 mL distilled water (dH2O) and a stir bar. Dissolve 80 g of NaCl, 2 g of KCl, 14.4 g of Na2HPO4, and 2.4 g of KH2PO4. Once in solution, adjust the total volume to 1 L with dH2O. Take 100 mL of 10× PBS, and dilute to 1 L with 900 mL of dH2O. Autoclave. 2.1.2 4% Paraformaldehyde/PBS Solution Weigh out 40 g of paraformaldehyde (PFA) and place in a beaker within a fume hood. Pour in PBS to a total volume of 1 L. Add a stir bar and put the beaker in an 80 °C water bath on a heated stir plate and stir solution. Once the temperature of the PFA solution equilibrates to that of the water bath, the vast majority of the PFA powder will go into solution. Filter the solution (in the fume hood) and aliquot into 50 mL screw cap tubes and store in the freezer. Thaw when needed. Once thawed, store the tube at 4 °C for up to 2 weeks. 284 2.2 Jeffery R. Barrow Probe Synthesis Reagents and equipment for probe synthesis: 1. Sterile dH2O (see Note 1a). 2. Sterile microcentrifuge tubes. 3. 3 μM (10×) stock of M13 forward primer: 5′ GTA AAA CGA CGG CCA GT. 4. 3 μM (10×) stock of M13 reverse primer: 5′ GGA AAC AGC TAT GAC CAT G. 5. 10× dNTP stock (1.25 mM for each nucleotide). 6. Taq polymerase (and associated 10× Taq buffer). 7. Thermocycler. 8. T3, T7, and Sp6 RNA polymerase (and associated 10× RNA polymerase buffers). 9. 10× DIG nucleotide labeling mix (Sigma); other labeling mixes can be used: FITC, DNP, or Biotin (Sigma). 10. 4 M LiCl solution. 11. 100% and 70% ethanol. 12. 0.7% agarose gels (prepared by dissolving 0.7 g of agarose in TAE or appropriate electrophoresis buffer). 13. Microcentrifuge. 2.3 In Situ Hybridization Day 1 Reagents/materials for dehydration and prehybridization: 2.3.1 PBST PBST is made by taking 100 mL 10× PBS and diluting with ~900 mL dH2O and 1 mL Tween-20. 2.3.2 Methanol Series 25%, 50%, and 75% methanol are made by diluting 100% methanol in PBST for 25% and 50% dilutions and in dH2O for 75% (salts in PBST will precipitate in high-percentage methanol solutions). The percentage is based on volume methanol/volume solution (e.g., a 25% solution is made by taking 25 mL methanol and adding 75 mL PBST, i.e., 25 mL methanol in 100 mL of total solution). 2.3.3 6% H2O2 Bleach Generally 3 mL of 6% H2O2 bleach is required per embryo in a Netwells® basket. For three baskets, approximately 10 mL of bleach (see Note 1b) will be required. One would therefore take 2 mL of 30% H2O2 + 8 mL PBST to make 10 mL of 6% H2O2 bleach. 2.3.4 Proteinase K Per 1 mL dH2O, take 10 mg of proteinase K and dissolve. The proteinase K should dissolve easily at room temperature. Once dissolved, distribute in 50 μL aliquots in microcentrifuge tubes and store at −20 °C. Do not freeze/thaw a given tube more than five times (which can be monitored by making a hash mark on the tube every time it is thawed). Netwells® trans-well baskets 15 mm diameter/74 mm mesh Costar catalog number 3477 (see Fig. 2). Whole-Mount In Situ Hybridization 285 Fig. 2 Netwells® trans-well baskets in a 12-well dish To make up 10 mL of 10 μg/mL proteinase K in PBST (see Note 1b), take 1 μL of 10 mg/mL stock of proteinase K, and mix in 10 mL of PBST. 3 mL per basket is adequate volume for protease treatment of embryos. 2.3.5 2 mg/mL Glycine/ PBST Take 24 mL PBST (see Note 1b) and add 48 mg of glycine. Mix by swirling or inversion until the powder dissolves (essentially instantaneous). 2.3.6 4% Paraformaldehyde/0.2% Glutaraldehyde Take 9 mL (see Note 1b) of PFA/PBS (prepared above), and add 72 μL of 25% glutaraldehyde. 2.3.7 20× SSC is made by dissolving 175.3 g NaCl and 88.2 g of sodium citrate into 800 mL of dH2O. Adjust pH to 4.5 with 1 M HCl. Add dH2O to 1 L. 20× SSC pH 4.5 Prehybridization (Prehyb) solution (28 mL; see Note 1b) 50% formamide 14 mL formamide 5× SSC pH 4.5 7 mL 20× SSC pH 4.5 dH2O 7 mL 1% SDS 0.28 g 50 μg/mL heparin 140 μL 10 mg/mL heparin/dH2O stock 50 μg/mL yeast tRNA 140 μL 10 mg/mL yeast tRNA/dH2O stock 286 Jeffery R. Barrow Hybridization (Hyb) solution is made by taking a volume of prehyb and adding 5 μL of RNA probe for each milliliter. 2.4 In Situ Hybridization Day 2 and 3 (See Note 1b) Make up three washes: Solution I 30 mL (2.5 washes for 3 baskets at 4 mL/wash) 50% formamide 15 mL formamide 5× SSC pH 4.5 7.5 mL 20× SSC 1% SDS 0.3 g 7.5 mL dH2O Solution II 66 mL total (5.5 washes) 0.5 M NaCl 6.6 mL of 5 M NaCl 10 mM Tris–HCl pH 7.5 0.66 mL of 1 M Tris–HCl 0.1% Tween-20 66 μL Tween-20 58.7 mL dH2O Solution III 24 mL (2 washes) 50% formamide 12 mL formamide 2× SSC pH 4.5 2.4 mL SSC 4.5 9.6 mL dH2O 2.4.1 10 mg/mL RNaseA Solution 2.4.2 10× Maleic Acid Buffer pH 7.5 (MAB) Take 10 mg of RNaseA and dissolve in 1 mL of dH2O. 1.0 M maleic acid 116 g Maleic acid 1.5 M NaCl 87 g NaCl 800 mL dH2O Adjust pH of the 10× MAB to 7.5 by the addition of NaOH (~40 g of NaOH pellets). Note: MAB is a weak buffer so the pH 7.5 endpoint is easily bypassed. After pH 7.5 is reached, add dH2O to 1 L. MBST is made by adding 100 mL 10× MAB and ~900 mL of dH2O and 1 mL of Tween-20. One should keep MBST at 4 °C to prevent microbial growth. 2.4.3 Antibody Pre-block Solution Make up 9 mL (see Note 1b) of 10% sheep serum pre-block: 900 μL of heat-inactivated sheep serum (HISS) (Sigma S2263) in 8.1 mL of MBST. Sheep serum is heat-inactivated at 70 °C for 30 min and then aliquoted and stored at −20 °C. Heat inactivation is important to inactivate endogenous alkaline phosphatase activity in the serum. Whole-Mount In Situ Hybridization 287 2.4.4 1:100 Antibody Stock Solution Make up 1 mL of 1:100 dilution of anti-DIG antibodies: take 890 μL of MBST, 100 μL of HISS, and 10 μL of anti-DIG antibodies (Sigma 11093274910). Store at 4 °C; see Note 1c. 2.4.5 1:5000 Anti-DIG Antibody Hybridization Solution To make up 10 mL (see Note 1b) of 1:5000 anti-DIG/10% HISS/ MBST: 180 μL of 1:100 diluted antibody stock. 900 μL of HISS. 8.9 mL pf MBST. 2.5 In Situ Hybridization Day 4 Color Reaction 2.5.1 NTMT Buffer NTMT 25 mL (see Note 1b) 100 mM NaCl 0.5 mL of 5 M NaCl 100 mM Tris–HCl pH 9.5 2.5 mL of 1 M Tris–HCl pH 9.5 50 mM MgCl2 1.25 mL of 1 M MgCl2 0.1% Tween-20 25 μL Tween-20 20.75 mL dH2O 2.5.2 BM Purple Reagent Reagent is purchased from Sigma (B3679). 2.5.3 PBS/EDTA Take 500 mL of PBS and add 2 mL 0.5 M EDTA pH 8.0. 3 Methods 3.1 Embryo Collection 3.2 DNA Template Synthesis For whole-mount in situ hybridization, the best results are obtained when embryos are younger than E13.5 mouse embryos (see Note 2a) and younger than HH 29 in chick embryos (see Note 2b). Embryos are collected in ice-cold PBS and then transferred to cold 4% paraformaldehyde (PFA) and allowed to fix in overnight at 4 °C. 1. PCR template synthesis: (see Note 3a) 5.0 μL 10× Taq buffer. 5.0 μL dNTPs (from stock containing 1.25 mM of each stock). 1.0 μL of 20 ng/μL plasmid template. 5.0 μL 3 μM M13 forward primer (assuming your plasmid has the sites). 5.0 μL 3 μM M13 reverse primer. 28 μL dH2O. 1.0 μL Taq polymerase 50 μL total. 288 Jeffery R. Barrow 2. PCR conditions/program: 2 min 95 °C. 30–35 cycles 1 min 95 °C. 30 s 54 °C. 2 min 30 s 72 °C. Following the 35 cycles: 10 min 72 °C. Hold at 4 °C. 3.3 Probe Synthesis 1. Set up transcription reaction in a microcentrifuge tube: 11 μL dH2O. 2 μL 10× Transcription Buffer (Sigma). 2 μL 10× nucleotide DIG labeling mix (other hapten nucleotide mixes can be used). 4 μL PCR template (~1 μg). 1 μL RNA polymerase (Sigma). 20 μL Note 3b. 2. Incubate transcription reaction at 37 °C 1.5–2 h. 3. Run 1 μL of the reaction via gel electrophoresis (0.7% agarose gel) to determine the robustness of the RNA probe synthesis. After removing a microliter, the reaction can be placed back at 37 °C during the time the gel is running or can be placed on ice or in the freezer. A strong RNA band should be observed (see Fig. 3). A robust synthesis should yield 10 μg of RNA (see Note 3c). 4. To stop the reaction and prepare for precipitation of the probe, add: 80 μL dH2O to the reaction. 10 μL 4 M LiCl. 300 μL ice cold, 100% ethanol. 5. Incubate at −20 °C for 1 h to overnight. 6. Spin for 30 min at 14,000 rpm (~20,000 × g) at 4 °C (this can be performed in a microcentrifuge stored at 4 °C). 7. Wash the pellet with 70% ethanol. 8. Spin 2 min at 14,000 rpm (~20,000 × g) at 4 or 25 °C; carefully remove 70% ethanol. 9. Air-dry 5–10 min. 10. Resuspend pellet in 100 μL of dH2O (~0.1 μg probe/μL). Store at −20 °C (see Note 3d). Whole-Mount In Situ Hybridization 289 Fig. 3 0.7% agarose gel exhibiting the electrophoresis of 1 kilobase ladder (1 kb ladder) and 1 μL each of 20 μL transcription reactions for chick Fgf8 (Fgf8) and chick Sonic Hedgehog (Shh) RNA probes. In both cases, the yield of the RNA appears to be robust. Further the nucleic acids are in good condition as evidenced by the sharp lower boundary of the band 3.4 In Situ Hybridization 3.4.1 Day 1: Hybridization 1. Remove PFA from embryos and rinse two times in PBST. 2. Dehydrate embryos through a methanol series 25% methanol/ PBST; 50% methanol/PBST; 75% methanol/dH2O; 2 × 100% MeOH for 10 min each. See Note 4b. 3. Embryos that are completely dehydrated can be stored in 100% methanol at −20 °C for short periods of time (< 1 month; thereafter in situ hybridization staining may diminish) or they can be immediately rehydrated for continuation of the protocol below. 4. Rehydrate embryos in 75% in dH2O, 50%, and 25% MeOH in PBST for 10 min each or until the embryos equilibrate (as evidenced by embryos sinking to the bottom of the tube or basket). 5. Rinse embryos two times in PBST for 10 min each at room temperature (RT). 290 Jeffery R. Barrow 6. Bleach with 6% H2O2 for 1 h at RT. 7. Wash three times in PBST for 5 min each at RT (4 mL of PBST per wash). 8. Treat with 10 μg/mL proteinase K in PBST for an appropriate time (see Note 4c). 9. Wash two times with fresh 2 mg/mL glycine in PBST for 5 min each at RT (see Note 4d). 10. Wash two times for 5 min each in PBST at RT. 11. Refix with 4% paraformaldehyde/0.2% glutaraldehyde in PBST/for 20 min at RT (see Note 4e). 12. Wash three times in PBST for 5 min each at RT. 13. Equilibrate in prehybridization (prehyb) solution for 5 min or until the embryo sinks. 14. Incubate in prehyb for 1–4 h at 68 °C. 15. Transfer to hybridization (hyb) solution overnight at 68 °C. Wrap cellophane to cover Netwells®, and place lid over the dish and then place in 68 °C incubator. 3.4.2 Day 2: Posthybridization Washes/ Antibody Hybridization (~6.5 h) Make up fresh Solutions I, II, and III. 1. Pre-warm Solution I to 68 °C. 2. Collect the hyb solution that embryos have been sitting O/N in a tube or bottle, and store at −20 °C (it can be reused at least five times). 3. Wash embryos twice for 30 min each in Solution I at 68 °C. 4. Prepare a 1:1 mixture by adding 6 mL of Solution I and 6 mL of Solution II and prewarm at 68 °C. 5. Wash embryos for 10 min in Solution I/II mixture at 68 °C. 6. Wash embryos three times for 5 min each in Solution II at RT. 7. Incubate embryos for 1 h in 100 μg/mL RNase A in Solution II at 37 ° C (prepared by adding 120 μL of a 10 mg/mL RNaseA stock in 12 mL of Solution II) (see Note 4f). 8. Wash embryos for 5 min in Solution II at RT. 9. Wash two times for 30 min each in Solution III at 68 °C. 10. Wash three times for 5 min each in MBST at RT. 11. Pre-block embryos in 2–3 mL of 10% sheep serum in MBST for 2.5 h at RT. 12. Transfer embryos to 2–3 mL of 1:5000 anti-DIG antibody hybridization solution. Wrap cellophane to cover Netwells®, and place lid over the dish and then incubate at 4 °C overnight (see Note 4g). Whole-Mount In Situ Hybridization 3.4.3 Day 3: Postantibody Washes 291 1. Wash embryos three times for 5 min each in MBST at RT. 2. Wash eight times for 1 h each in MBST at RT. 3. Transfer into a new well of MBST and incubate overnight at 4 °C. 3.4.4 Day 4: Color Reaction 1. Take BM Purple out of the refrigerator so that it can equilibrate to RT. 2. Incubate the embryos two times for 20 min each in 3 mL of fresh NTMT (see Note 5a). 3. Mix the BM Purple solution by inversion of the bottle two or three times. Pipet 2–3 mL of BM Purple to each well. Transfer the baskets to the BM Purple. 4. Incubate the embryos in the dark at RT for about 2 to 6 h, monitoring the purple signal from time to time (i.e., every hour; Note 5b). For some probes, the incubation time may be significantly longer. 5. To temporarily stop or slow down the reaction (if necessary), embryos can be placed back in NTMT at 4 °C. Embryos can then be replaced in BM Purple to resume the reaction. 6. The color reaction can be completely arrested by storing the embryos in vials of PBS + 2 mM EDTA. Embryos can be stored indefinitely at 4 °C in vials of PBS/EDTA. See Note 5c. 4 Notes 1. Materials (a) Many in situ protocols mention the use of diethyl pyrocarbonate (DEPC) to inactivate RNases in water or aqueous solutions. I have found DEPC treatment to be unnecessary for the overall success of the procedure. (b) Volumes of solutions to be prepared are calculated based on what is required for three Netwells® baskets. (c) Some protocols suggest subtraction of the anti-DIG Fab fragments (Sigma) with mouse or chick embryo acetone powder. I have found this step to yield minimal benefit, if any, with these antibodies. 2. Embryo Staging (a) Mouse embryos are staged as previously described [24]. Briefly, it is assumed that fertilization takes place at midnight previous to the day that a vaginal plug is identified in a female mouse. At noon the following day, the embryos are said to be at embryonic day (E) 0.5 or 0.5 days post coitum (dpc). Therefore, on midnight 10 days following 292 Jeffery R. Barrow the presumed fertilization, embryos are said to be at E10/10 dpc, and at noon later that day, they are at E10.5/10.5 dpc, etc. (b) Chick embryos are staged according to a series of chronological, morphologic criteria described by Hamburger and Hamilton [25]. For example, for Hamburger and Hamilton (HH) stages 1–6, the shape and length of the primitive streak are used for staging the embryos, whereas neural tube morphology (HH 5–8), somite number (HH 8–14), limb morphology (HH 15–35), etc. are used for subsequent staging criteria. 3. Probe Synthesis (a) A linear DNA template containing a cDNA (or portion thereof) of a gene of interest and a promoter on the 3′ end of this template is required to make an antisense RNA probe. This linear sequence can be generated by restriction enzyme digestion of the circular plasmid at a unique position 5′ to the cDNA sequence. Alternatively, a linear sequence can also be generated by PCR amplification using M13 forward and reverse primers which in most cloning plasmids immediately flank a cDNA insert. Given that the PCR-generated fragment is almost entirely composed of template sequence for the in situ probe, it is superior to templates that have been linearized via restriction enzyme digestion (which would also contain plasmid sequence). (b) Some protocols recommend the addition of RNase inhibitor; however, I have not found this treatment to be necessary. (c) Some protocols recommend digestion of the DNA template with DNase I for 15 min prior to precipitation. I have not found this step to be necessary. (d) If a lower yield is observed based on the intensity of the RNA band, resuspend in less than 100 μL of dH2O to approach ~0.1 μg probe/μL. 4. In Situ Hybridization (a) In situ hybridization is a procedure that requires many solution changes over the course of the protocol. It is often cumbersome (especially with very small embryos) to remove and replace solutions. A very convenient means to change solutions is to place embryos in Netwells© baskets (Corning, see Fig. 2) and transfer the baskets from 1-well in a 12-well dish containing an appropriate solution or wash to the next. Generally speaking, one must put in a minimum of 2–2.5 mL of a given solution in a well to ade- Whole-Mount In Situ Hybridization 293 quately cover the embryos (i.e., when trying to conserve a solution such as probe or antibody solutions) and a maximum of 4 mL (i.e., for washes when larger volumes are desired). An approach that works well is to place the basket in the first well of a column of 4 in a 12-well dish. One transfers the basket from one well to the next, down a column then back to the top, completely removing solutions after incubations and replacing with appropriate solutions/washes according to the sequence in the protocol. One can run three baskets simultaneously per dish. All incubations and washes are performed in stationary manner—no rocking is required. (b) It is critical to completely dehydrate embryos to avoid the formation of bubbles during the H2O2 bleaching step. Embryos at E10.5 or younger can be adequately dehydrated using the Netwells® baskets at 4 mL per wash. For larger embryos (E11.5 or older), it is important to dehydrate in larger volumes (i.e., 8–10 mL of 100% methanol in a 15 mL conical tube) and for longer times (15 min). (c) Proteinase K treatment is one of the most critical steps of the protocol. If not treated long enough, probe will only penetrate the most superficial tissues. If treated too long, the tissue of interest (or the entire embryo) might be digested away. The extent of time of proteinase K treatment depends on the age of the embryo and the tissue being examined. Suggested times: Time in 10 μg/mL proteinase K at 25C (min) Mouse (E) Chick (HH) E7.0–7.5 HH 4 and younger 2 E8.5 HH 5–6 5 E9.5 HH 7–15 15 E10.5 HH 16–21 25 E11.5 HH 22–26 35 E12.5 HH 27–29 45 These times are recommendations. As activity of proteinase K can vary between lots and preparations, it is important to optimize digestion times. It is important to note that if one is looking at expression in surfac e ectodermal structures (e.g., the apical ectodermal ridge), times will be very short even in older embryos. 294 Jeffery R. Barrow (d) Glycine in the PBST wash quenches proteinase K to immediately stop digestion. (e) After proteinase K treatment, embryos become quite fragile. Refixation in PFA/glutaraldehyde reinforces the embryos. (f) RNaseA treatment reduces background by digesting any single-stranded (non-hybridized) RNA probe. (g) The anti-DIG antibody binds to the RNA probe (via digoxigenin-modified uridine residues). The antibody is conjugated to alkaline phosphatase which will provide the basis to indirectly visualize the RNA probe. 5. Color Reaction (a) The purpose of the NTMT pre-washes is to equilibrate the embryos to an alkaline pH and divalent cation (Mg2+) so as to promote the best conditions for the alkaline phosphatase enzyme that is conjugated to the anti-digoxigenin antibody. (b) In cells where the alkaline phosphatase-conjugated anti-DIG antibodies have bound, the alkaline phosphatase converts the BM Purple substrate to a dark purple precipitate. (c) EDTA chelates divalent cation, effectively blocking the activity of the alkaline phosphatase. The alkaline phosphatase activity can also be abrogated also by 15 min of postfixation with 4% paraformaldehyde/0.1% glutaraldehyde. If alkaline phosphatase activity is not inactivated, the entire embryo will turn a deep purple within several weeks when stored at 4 ° C in PBS. References 1. 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