High-throughput holistic in situ hybridization for the detection of environmental disturbances in zebrafish (Danio rerio), an embryonic tissue-specific gene expression alteration experiment
High-throughput holistic in situ hybridization for the detection of environmental disturbances in zebrafish (Danio rerio), an embryonic tissue-specific gene expression alteration experiment
Holistic in situ hybridization is a process that allows observation of cellular gene expression (mRNA) in intact organisms. By comparing differences in gene expression regions of organisms in different environments, it helps us to understand the cell- and tissue-specific effects of environmental exposures. This technique complements existing gene expression profiling techniques, such as DNA microarrays, which can only reflect changes in gene expression levels in a single organism or tissue.
By Martin, this experiment is from "Environmental Genomics Lab Guide".
Operation method
High-throughput holistic in situ hybridization for the detection of environmental disturbances in zebrafish (Danio rerio), an embryonic tissue-specific gene expression alteration experiment Move I. Materials All solutions must be prepared with RNase-free reagents and DEPC-treated distilled water. Instruments in contact with embryos, instruments, tools and reagents in contact with embryos are subject to RNase inactivation (see Note 1). (1) Heat block for making in situ hybridization baskets. (2) In situ hybridization basket: Remove the tip of the 1.5 m L centrifuge tube or other cylindrical object and place a piece of fine nylon in the basket. Remove the tip of the 1 m L centrifuge tube or any other cylindrical object, and attach a piece of fine nylon mesh to the remainder of the centrifuge tube by low-temperature melting using a heating plate. Remove the excess nylon mesh. Note Careful not to overheat or you will melt the nylon mesh. (3) Six-hole plate (BectonDickinson, cat. no. 1146). (4) Flat-tipped forceps for moving the in situ hybridization basket between grid wells. (5) Glass pipette for moving the embryos. (1) f X g purified linear plasmids containing the target gene (see Note 2). (2) IOXDIG labeling mixture: 10 mmol/L A T P, 10 mmol/L C T P, IOmmolZLGTP,6. 5 mmol/L UTP, 3. 3 mmol/L DIG-11UTP (Roche)0 (3) Transcription buffer and bovine serum albumin (BSA)/dithiothreitol (DTT; isoenzymes together). (4) Phenol-chloroform-isoamyl alcohol (PCI) : Phenol-chloroform-isoamyl alcohol was mixed in the ratio of 25 : 24 : 1 and stored at 4°C in the dark. (5) Chloroform-isoamyl alcohol (CIA): chloroform-isoamyl alcohol mixed in the ratio of 24 : 1, stored at 20°C. (1) 4% PFA-PBS solution: 80 mL of 1XPBS containing 4% paraformaldehyde. slowly add NaOH until paraformaldehyde is completely dissolved. adjust p H with H Cl to 7.2. add distilled water (dH20) to 100 mL. store at 4°C for short-term storage (1~20 weeks). Adjust p H with H Cl to 7.2. Add distilled water (dH20 ) to 100 mL. Store at 4°C for short term (1 to 2 weeks) and at _20°C for long term. d (2) 5XPBS: 40 g NaCl, I g KC1, 14.4 g Na2 HPO4, 2. 4 g KH2PO4, add DEPC-treated water to 800 mL, adjust pH with H Cl to 7. 4. Add DEPC-treated water to a final volume of I L. Dilute to 1XPBS. (1) PBSTween (PBST): 0.1% Tween-20, 1XPBS. (2) Protease 1<: (50 nets /11^): Dissolve 50 11^ of Protease 1^ powder in water, dispense and store at 20°. (3) Glycine solution: Prepare 2.7 mmol/L glycine with IX PBS. (4) 5X pre-absorbed anti-DIG antibody solution: anti-DIG antibody (Roche) diluted at 1:10,000, 2% calf serum, 2 mg/mL BSA and 20-50 embryos fixed in IX PBST, let the solution stand at room temperature for I h or overnight at 4°C. Store at 4°C and dilute to IX with PBST when used (see Note). Store at 4°C and dilute to IX with PBST at the time of use (see Note 3). (5) 20XSSC: 3 mol/L NaCl, 0.3 mol L sodium citrate, pH 7. Autoclave. (6) Hybridization solution: 50 % formaldehyde, 5 X SSC, 0.1 % Tween-20, 50 pg/mL heparin, 100 pt/mL heparin. 1 % Tween-20, 50 pg/mL heparin, 100 ptgZmL Enzyme tRNA, 9 mmol/L citric acid, stored in a 20. (7) Post-hybridization mix (post-hyb mix): 50 % methyl alcohol, 5 XSSC, 0.1 % Tween-20, 50 fig/m L heparin, 9 111111.1,/1 citric acid. (7) Post-hybridization mix (post-hyb mix): 50 % methylamine, 5 XSSC, 0.1 % Tween-20, 50 fig/m L heparin, 9 111111 〇 1,/1 citric acid, stored at 20 °C. (8) 100 ng probe (1 to 2 p L suspension probe) (see 3.1). (9) Sequestering solution: 2 % calf serum, 2 mg/m L BSA, add to final volume with PBST. (10) Staining buffer: 100 mmol/L Tris-HCl, pH 9.5, 50 mmol/L MgCl2, 100 mmol/L NaCl, 0-1 % Tween-20, 0.4 mmol/L Ievamisol (see Note 4). (11) NBT storage solution: 70 mg/mL nitrobluetetrazolium (NBT) dissolved in dimethylformamide, stored at 4°C, protected from light. (12) BOP stock solution: 50 mg/mL p-toluidine blue (5-bromo-4-chloro-3-indolyl phosphate, 5-bromc^ - chloro(1)-3-indolyl, BOP) dissolved in 70 % dimethylformamide, stored at 4°C protected from light (see Note 5). (13) Embryo preservation solution: contains 0.025 % sodium azide. 0.25 % sodium azide in 1XPBS. (1) Prepare a DNA template by digesting 10 Kilograms of plasmid DNA (containing the target sequence) with an appropriate endonuclease according to the instructions. The endonuclease should be cleaved at the 3' end of the gene sequence to synthesize an antisense probe and at the 5' end to synthesize a positive control probe (see Note 6). (2) Templates can be extracted by commercial purification column systems or by mixing equal volumes of PCI and CIA. (3) The extracted template is further precipitated with 1/10 volume of 3 mol/L sodium acetate and two times the volume of 100 % ice ethanol. (4) The solution was gently shaken and centrifuged at maximum speed for 30 min. (5) The DNA precipitate is washed with 75 % cold ethanol, air-dried and dissolved in 10 juL of DEPC water. (6) Quantify the concentration of template by spectrophotometry or agarose gel electrophoresis with standards. (7) Probe synthesis is required by the following reactions, the total volume of which should not exceed 20 . The probe synthesis reaction consists of l Mg of linear DNA template, IX DIG Labeling Mix, IX Transcription Buffer (with transcriptase), DTT or BSA (with transcriptase), 40 U of RNasin (Promega), and 20 to 50 U of RNA polymerase. Select the appropriate temperature and time for incubation (usually I h) depending on the RNA polymerase used. (8) Add another 20~50 U of RNA and incubate with the polymerase for I h. (9) Add 40 U of DNase ]; incubate at 37°C for 10 min (see Note 7). (10) Add a final concentration of 0.02 mol/L EDTA, 0-5 mol/L LiCl, 70% ethanol, and incubate at 8(TC) for 1~2 h or overnight to precipitate the probe. (11) Centrifuge at 14°C for 30 min at maximum speed to separate the probes. (12) Wash the probe precipitate with 99% ethanol, air-dry, and re-dissolve in 50/xL or less DEPC water and store at _80°C. 2 pL of the probe is aspirated onto an EB-stained agarose gel for electrophoresis. Determine the quality of the probe by electrophoresis of 2 pL on an EB-stained agarose gel. Unless otherwise indicated, all embryo manipulations were performed in homemade small baskets placed in single wells of a six-well plate (Fig. D . --Six baskets can be placed in a single well of a six-well plate, and the baskets are transferred between wells containing different incubation solutions using flat-tip forceps. (2) Lacquered embryos were washed with IX PBS before dehydration and stored in methanol at 20°C. (3) Store embryos in methanol at least overnight before performing in situ hybridization. Embryos can be stored in methanol at 20°C for several years. (1) Select embryos and rehydrate by incubation in the following solutions: 7 5% MeOH-25% PBS, 50% MeOH-50% PBS, 2 5% Me 〇 H-75% PBS for 5 min each; incubate with PBST 4 times for 5 min each time. rehydrated embryos can be used for in-situ hybridization and the preparation of pre-absorbed antibody solution. For the preparation of pre-absorbed antibodies, refer to 2. 4. 4. (2) Experimental embryos 24 h or older are treated in PBST containing proteinase K (0.1 nets/mL) for a predetermined time (see Note 9). (3) Embryos were rapidly washed with PBST and incubated for 5 min in PBST containing 2 mg/m L glycine. (4) Embryos were fixed with 4% PFA-PBS for 20 min and washed with PBST. (5) Pipette the embryos into I.5 mL centrifuge tubes and prehybridize them for I h at 65°C in hybridization buffer. (6) Hybridize overnight at 65°C in fresh hybridization solution spiked with 1~2/100 fzL probe. (7) Pipette the embryos into small homemade baskets. (8) Embryos were incubated for 10 min each at 65°C in the following solutions: . 7 5 % post-dye-crossing mixture-25% 2XSSC 50 % Post-dyeing mix - 50% 2XSSC 2 5 % Post-dyeing mixture - 75% 2XSSC 100% 2X SSC (9) Embryos were washed twice in 60°(:, 0.2\33 〇 for a total of 30 111 丨 11 . Embryos were then washed at room temperature in the following solutions incubated for 5 min each in the following solutions. 7 5 % 0. 2XSSC-25% PBST 5 0 % 0. 2XSSC-50% PBST 2 5 % 0. 2XSSC-75% PBST 100% PBST (1) Embryos are incubated for 1 to 4 h at room temperature in a containment solution, and then incubated for 3 to 4 h at room temperature in pre-absorbed anti-DIG antibody (1:5000). (2) The embryos are washed 6 times with PBST for 15 min at room temperature to remove excess antibody (see Note 10). (3) Embryos are pre-incubated with staining buffer for 5 min at room temperature. (4) Staining reactions were performed in staining buffer containing 0 -4 mmol/L N BT and 0. 4 mmol/L BCIP. The staining reaction time depends on the probe and the expected gene expression. Staining reactions should be observed every 15-30 min to determine the appropriate staining time. Embryos in small baskets can be viewed with a standard stereotaxic microscope. The staining procedure should be done in the dark, avoiding light as much as possible. The longer the embryos are placed in the staining mixture, the stronger the staining; however, if the staining time is too long, the background will be very strong, which makes it difficult to distinguish between true staining and background, especially if the expression level of the gene being tested is very low (see Note 11). (5) When the staining is completed, the embryos are washed twice with PBST for 15 min at room temperature. (6) The embryos were then fixed with 4% PFA at room temperature for 2 h or overnight at 4°C. (7) Embryos are washed with PBS and stored in PBS containing 0.025% sodium azide at 4°C for long term storage. The following automated procedure uses the Insitu P ro automated in situ hybridization system developed by Intavis Bioanalytical Instruments AG (Koeln, Germany). The embryos are rehydrated and treated with proteinase K before entering the instrument (see step 3. 3. 3), and the staining is done manually (starting from step 3. 4.4). The procedure begins with washing the apparatus at room temperature (TO [OFF]). The embryos are then incubated in 125 Hybridization Buffer (C) for 20 min at room temperature (step 5), the temperature is raised to 65°C (T 2 [HIGH]) and then incubated in fresh Hybridization Solution for 90 min. The probes are added to the embryos and incubated for M h at 65°C (step 8). Embryos were washed for another 20 min with 150 of each of solutions D, H, F, and E, which have different concentrations of SSC and hybridization solution (steps 9 to 13). After the temperature is lowered to room temperature again (TO [OFF]), the embryos are washed for 20 min each with 150 of solutions I, J, K, and A. After 60 min of incubation in 120 of solution L (blocking solution), the embryos are incubated in 120 of solution M (DIG antibody) for 4 h. The program waits for a user command after the embryos have been washed seven times for 20 min each with 150 of solution A (PBST) (step 30, wait for NTMT to start). The program waits for a user command (step 30, wait for NTMT). The user removes the embryos from the instrument and proceeds directly to step 3. 4. 4. The instrument is automatically cleaned at the end of the program. This program can process 500 embryos in 48 hours. The containers used in the Insitu Pro System and the solutions in them are specifically labeled as follows (see Note 12). A : PBST B: 50 % Methanol C: Hybridization buffer D: 75 % hybridization buffer + 25 % 2XSSC E: 0.2XSSC F: 2XSSC H : 25 % hybridization buffer + 75 % 2XSSC I : 7 5 % 0. 2 XSSC+ 2 5 % PBST J: 50 % 0.2 XSSC + 50 % PBST K: 25 % 0.2 XSSC + 75 % PBST L : Locking solution M : DIG antibody Probe: DIG labeled RNA probe The program codes are listed below: Quantification of the in situ hybridization signal is achieved by means of a digital optical microscope and relatively basic image analysis software. Digital images are taken of the processed embryos and saved in a format recognized by the image analysis software. To ensure that the data are acquired without human bias,' imaging and analysis should be done in a double-blind manner. In addition, it should be ensured that all embryos are in a similar arrangement prior to image acquisition to avoid changes in the data obtained from the images due to changes in viewing angle. Therefore, the embryos can be fixed on a petri dish lined with a layer of agar prior to photography. Using small forceps or a pipette, small grooves or holes are dug in the gel in which the embryos can be placed to ensure a fixed orientation. Finally, a consistent light intensity is applied to ensure that the color values of the in situ hybridization signal are as uniform as possible from sample to sample. After obtaining an image of the embryo, the image can be analyzed using any of a number of image analysis software packages, including AdobePhotoshop (www.adobe,com), NIH Image (http://rsb.info.nih.gov.nih-image), Sim pie PCKhttp: //www. cimaging.net/ ) ^; Metamorph (http: '^ oleculardevices.com/pages/software/metamorph.html). All of these software programs are capable of determining various image parameters (area, length, density, etc.) and in most cases outputting the data into a recognizable format, such as a spreadsheet, for subsequent use. Caveat 1. Dry solid reagents must be packaged unopened and used only for RNase-free operations. Most dry reagents are available for purchase without RNase. DEPC water is prepared by adding I mL of DEPC to 1000 mL of distilled water. The solution must be vigorously shaken to dissolve the DEPC and allowed to stand for I h before being autoclaved and cooled for use. Alternatively, purchase prepared DEPC water. Storage bottles and other utensils should be treated with an RNase detergent such as RNA Zap (Ambion). 2. Plasmids for in vitro transcription of RNA probes must contain an RNA polymerase promoter site such as T7, T3, or SP6, e.g., pCR2 (Invitrogen) or Bluescriptn (Stratagene). 3. Embryos used for the preparation of pre-absorbed antibodies should be at the same stage of development as the embryos for which in situ hybridization is performed. In addition, the embryos may be chopped or crushed and added to the solution. At this point, the preabsorbed antibody must be allowed to stand for 24 h or 9 - Proteinase K activity varies from source to source. It is necessary to test each batch of proteinase K stock solution for activity to determine the optimal incubation time. Because of their fragility, 24 h embryos have shorter incubation times (Im in) than 48 h embryos (2 to 4 min) and adult tissues (5 to 10 min). If the embryo is incubated in Proteinase K for too long, the embryo will disintegrate easily. Insufficient incubation time can also result in a sharp decrease in the staining signal. 4. Levamisole storage solution should be prepared fresh. Levamisole reduces the alkaline phosphatase activity in the embryonic tissue and thus reduces the background staining. But it also reduces the overall efficiency of the staining process. So if the background color is not very high, levamisole can be left out. 5. Avoid exposure of these reagents to light. 6. Avoid leaving a 3' protrusion after enzymatic digestion. This protrusion can significantly reduce the efficiency of the in vitro transcription reaction. 7. In some cases, adding DNase after probe synthesis can reduce the background. The DNase here should be strictly RNase-free.This step can be omitted if the background color is not very high. 8. Often, when probes are detected in a gel, a faint RN A fragment is observed. These probes can still be used, but probes with only one band work best. 9. Proteinase K activity varies from source to source. It is necessary to test each batch of Proteinase K stock for activity experimentally to determine the optimal incubation time. Because of their fragility, 24 h embryos have shorter incubation times (Im in) than 48 h embryos (2 to 4 min) and adult tissues (5 to 10 min). If the embryo is incubated in Proteinase K for too long, the embryo will disintegrate easily. Insufficient incubation time can also result in a drastic decrease in staining signal. 10. If desired, embryos can be placed in PBST overnight at 4°C. 11. 11. Embryos should be stained purple. If after approximately 3 h the embryos have not been stained, the staining process should be repeated using fresh staining solution and BCIP-NBT. Embryos can be placed in the staining solution at 4°C overnight. 12. The volume of solution required should be calculated based on the number of samples ready to enter the instrument. For more product details, please visit Aladdin Scientific website.
(1) 4 % P F A fixation of embryos with exfoliated membranes, 4°C overnight.
analysis. Pixel thresholding is used to automatically identify cells with in situ hybridization signals. After selecting a specific spectral color (usually blue in the case of NBT/BCIP stains) using the image software, the software itself determines the measurement value based on the selected pixel. Figure 2 shows the results of the analysis of zebrafish embryos after exposure to cadmium chloride. To determine whether cadmium exposure had an effect on embryonic brain development, we selected a probe that was expressed only in a diffuse region demarcating the midbrain and hindbrain for in situ hybridization. By analyzing 100 control and 100 treated embryos, and determining the expression of the probe in the midbrain and hindbrain, we were able to determine whether cadmium exposure had an effect on embryonic brain development.
area of the expression domain, we found that cadmium exposure leads to a significant enhancement of the expression of this gene within this tissue. 
