将滋养层和内细胞群分别染色，TUNEL检测牛胚胎凋亡

Differential staining of trophectoderm and inner cell mass in combination with TUNEL labeling of bovine embryos将滋养层和内细胞群分别染色，TUNEL检测牛胚胎凋亡

Simple protocol for differential staining/TUNEL analysis of bovine embryos

 The procedure for differential staining of trophoblast and inner cell mass is based on a protocol obtained from D.J. Walker and G.E. Seidel, Jr. at Colorado State University.  The procedure for TUNEL staining is adapted from as commonly used protocol in our laboratory.

 Materials

1)      PBS/PVP � Add 1 mg/mL polyvinylpyrrolidone (PVP) to 10 mM PBS

2)      4% paraformaldehyde � Obtain 8% paraformaldehyde from Electron Microscopy Sciences (#15710-SP) and dilute 1:1 with PBS.  Make fresh on the day of use and discard any leftover after using that day.

3)      0.5% Triton X-100 � Dilute Triton X-100 in 10 mM PBS to obtain a final concentration of 0.5% (v/v).  Make fresh on the day of use.

4)      Propidium Iodide (PI solution) - Prepare a 2.5 mg/ml stock 1 solution by dissolving PI (Sigma P4170) in PBS and store at 4°C.  On the day of use, dilute the stock 1 solution 1:25 in 0.5% Triton X-100 to give a working concentration of 100 µg/mL.  Discard whatever is not used on the day of use.   

5)      Hoescht 33258 (H Solution) - Prepare Stock 1 by dissolving 25 mg Hoechst 33258 (Sigma B2883) in 2.5 ml of distilled water (10 mg/ml). Store at 4°C.  On the day of use, prepare Stock 2 by diluting the Stock 1 solution 1:1000 in 4% paraformaldehyde to give a working concentration of 10 µg/mL.  Discard whatever is not used on the day of use. 

6)      Grid plates � Purchase from Fisher Scientific (#08-757-149)

7)      Na Citrate/Triton X Solution � Dilute Na Citrate in PBS to obtain a final concentration of 0.1% (w/v).  On the day of use, add 0.1% (v/v) Triton X-100 and discard whatever is not used that day.

8)      TUNEL kit � Purchase from Roche (#1684795) and store at -20°C.

9)      DNase � purchase RQ1 RNA free DNase (1 U/µL) from Promega (#610A).  Store at -20°C.  On the day of use dilute 10 µL DNase in 190 uL PBS to obtain a working concentration of 50 U/mL.

10)  RNase � Obtain RNase A (heat treated, 100 mg/mL) from Qaigen (#1901).  On the day of use dilute the RNase 1:2000 with PBS to give a working concentration of 50 µg/mL.

11)  Microscope slides � Fisherbrand Superfrost slides (#12-550-14). Coat slides with a 1:10 dilution of poly-l-lysine (Sigma #P8920) in distilled water.

 

Differential Staining Procedure

 

1)      Prepare 50 µL droplets of the PI solution on a grid plate.  Make enough droplets for each of your treatments.  TIP - It will help to make a circle on the plate using a PAP pen when making droplets with solutions containing Triton X-100 or for wash droplets following incubation in a Triton X-100 solution.

2)      Place the search plate on a slide warmer set to 39°C and allow the PI solution to warm up prior to beginning the staining procedure.

3)      TIP - conduct all following steps in a dark room to prevent bleaching

4)      Remove blastocysts from culture in as little medium as possible and place into the droplets of PI solution for 30 seconds.

5)      Wash embryos 3 times by transferring between three 50 µL droplets of PBS/PVP.

6)      Place embryos into a 50 µL droplet of the H solution and incubate at room temperature for 15 minutes.

7)      Wash embryos as described in step 3.

 

TUNEL Procedure

 

8)      TIP � If you wish to complete the TUNEL procedure at a later time then you may store your differentially stained embryos at 4°C in PBS/PVP in a foil covered dish for up to a week before doing the TUNEL procedure.

9)      Permeablize embryos in a 50 µL droplet of Na Citrate/Triton X solution for 10 minutes at room temperature.

10)  Wash embryos as described in step 3.

11)  Determine the number of µL of the TUNEL reaction mixture you will need.  To do this,   multiply the number of treatments you have by 25 and add 50.  This will give you enough to incubate all of your treatments plus the negative control.

12)  Prepare the DNase as described in the materials section for the positive and negative control embryos.  Next, prepare the TUNEL reaction mixture.  First, remove 50 µL of the label solution (tube #2), place into a microcentrifuge tube and return to -20°C until step 16.  Next, dilute the enzyme solution (tube #1) in a microcentrifuge tube 1:10 with the label solution (tube #2) to give the volume of reaction mixture that was calculated in step 11.  Return the leftover enzyme and label solutions, if there are any, to -20°C and place the date on top of each tube.

13)  Incubate the positive and negative control embryos in a 50 µL droplet of DNase and your sample embryos in 25 µL droplets of the TUNEL reaction mixture at 39°C for 30 min.  TIP - For this step make sure embryos are placed in a humidified container (a plastic box with a damp paper towel will be adequate) to prevent evaporation of the droplets. 

14)  Place the remaining TUNEL reaction mixture at -20°C until step 16.

15)  After step 13, wash embryos as described in step 3.

16)  Remove the label solution and the remaining TUNEL reaction mixture prepared in step 12 from the -20°C freezer.  Prepare the RNase solution as described in the materials section.

17)  Incubate negative control embryos in a 25 µL droplet of label solution and positive control embryos in a 25 µL droplet of the TUNEL reaction mixture.  For sample embryos, incubate them in 50 µL droplets of the RNase solution.  All incubations should be done at 39°C for 30 min in a humidified box.

18)  Wash positive and negative control embryos as described in step 3.  DO NOT WASH SAMPLE EMBRYOS.

19)  Place embryos onto slides in a small volume of glycerol. 

20)  Mount a coverslip over the slides and place in a dark box at 4°C until viewing with an epifluorescence microscope.  View slides within 1-2 days.

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