TITLE: Tissue collection 
AUTHORS: Gabriel J. J.  Barrero, Oksana  Polesskaya, Abraham  Palmer

[DESCRIPTION]
This protocol describes how to dissect a rat to collect various tissue from an animal. Collected tissue is intended to be used for phenotypic characterization of various traits. Tissues also can be used for DNA extraction

[STEPS]
SECTION: Setting up
1. Setting up 
SECTION: Setting up
1.1. Animals are fasted ~840 min 960 min (960 min), water should be provided.

SECTION: Setting up
1.2. .Prepare labelsfor tissue collection tubes/vials. All nesessary files are in “Barcoding package” folder.
SECTION: Setting up
1.3. Take the rats which are going to be processed this day and scan their RFIDs to prepare the file for tissue collection. 
SECTION: Setting up
1.4. Read the RFIDs into RFID worksheet (the yellow area) of the barcodes-text-ratGWAS.xlsx. (No inputting of a #-sign or ‘ necessary, the sheet is formatted as text and will automatically interpret everything as text). The program will enter five identical barcodes to the each row on the next sheet named “Barcodes for printing”. Five identical barcodes are printed in each row.
SECTION: Setting up
1.5. Print labels with barcode and RFID number using the Barcodes for printing worksheet and the Printing Cryo Babies.docx instructions. Print each sheet 6 times to get the 30 identical barcoded labels you need. 
SECTION: Setting up
2. Label freezer boxes (2”, 3”, or 5” high) with the Center (MI, TN, NY), type of tissue, the box number, and the date (e.g.TN-Batch 08 Feb-17).
SECTION: Setting up
3. Prepare containers for tissue collection.
SECTION: Setting up
4. Prepare abucket with dry ice with a glass beaker filled with isopenten for flash-freezing tissue.
SECTION: Setting up
5. Have a computer ready for data entry. Create a new file for each batch using a template RatSampleCollection_BatchNN_MI_DDMonYYYY_Template_v3, e.g. RatSampleCollection_Batch02_MI_04Jun2015.xlsx, for data entry. When collecting tissues from a rat, read the rat’s RFID again (keep cursor on the yellow cell B2 on the worksheet “Check numbers”). Then read the barcodes on the tubes to check whether they are readable and match (cursor position B3, B4...), the program gives a warning if the numbers don’t match.
SECTION: Glucose Measurement
6. Glucose Measurement
SECTION: Glucose Measurement
6.1. Put a test strip in the glucose meter (man by Contour NEXT)
SECTION: Glucose Measurement
6.2. Test the meter using the control solution; make sure the reading is within the range (ranges listed on the bottom of the strip box).
SECTION: Glucose Measurement
6.3. Put a test strip in the glucose meter.
SECTION: Glucose Measurement
6.4. Restrain the rat. Cut off ~1mm of the tip of the rat’s tail. Gently milk/stroke the rat’s tail from the base near the butt to the tip of the tail. Discard the first drop of blood.
SECTION: Glucose Measurement
6.5. Place a drop of blood on the glucose strip. Record the reading.
SECTION: Glucose Measurement
6.6. Note: if glucose measurement is collected at a different time than sacrifice, create a new row in the RatSampleCollection...xlsx spreadsheet, record the data into columns A through H, and copy “NA” into every other cell of that row.
SECTION: Weight
6.7. Take the weight in grams, at a 0.1 g precision. 
SECTION: Fecal pellet collection
7. Fecal pellet collection
SECTION: Fecal pellet collection
7.1. Place animal securely on the researcher’s arm. Assess if there are pellets in the rectum by gently feeling the rectal canal (externally, gently pinch the anus between index finger and thumb and massage gently back 1-2cm from the anus). If pellets are present, they will feel like small solid masses (1cm in length). 
SECTION: Fecal pellet collection
7.2. The pellet is gently pushed towards the anus, and immediately placed in the sterile tube, by holding the tube in position immediate to the anus.
SECTION: Fecal pellet collection
7.3. The pellets are snap frozen on dry ice and stored at-80 °Cfor later processing. This procedure should be performed as rapidly as possible to ensure integrity of the microbiota.

SECTION: Fecal pellet collection
7.4. If pellets are not present, repeat pellet extraction in a few minutes. If still no pellet, do not collect it and proceed to anesthesia.
SECTION: Isoflurane Anesthesia
8. Isoflurane Anesthesia 
SECTION: Isoflurane Anesthesia
8.1. Turn oxygen on to about 1L / minute.
SECTION: Isoflurane Anesthesia
8.2. Place rat in induction chamber that has oxygen and isoflurane lines attached to it.

SECTION: Isoflurane Anesthesia
8.3. Turn on the isoflurane at about 5%.
SECTION: Isoflurane Anesthesia
8.4. Wait a few minutes until the rat is lying down and eyes are slightly shut. 
SECTION: Isoflurane Anesthesia
8.5. Take cover off container and pinch the toes to ensure surgical plane of anesthesia.If rat reacts, put it back in the chamber for a little bit longer.If no reaction, take the rat out.
SECTION: Length
9. Length
tape the ruler to the table so it doesn’t move around.
SECTION: Length
9.1. 
Head to butt
-Measure in cm the length from tip of the nose to the end of the butt (0.1cm precision).

SECTION: Length
9.2. Head to tail
-Measure in cm the length from tip of the nose to the end of the tail. (0.1 cm precision)
SECTION: Blood Collection via Cardiac Puncture (NY, TN)
10. Blood Collection via Cardiac Puncture 
SECTION: Blood Collection via Cardiac Puncture (NY, TN)
11. Cutting open 
SECTION: Blood Collection via Cardiac Puncture (NY, TN)
11.1. Place the rat on its back on the table.
SECTION: Blood Collection via Cardiac Puncture (NY, TN)
11.2. Place the isoflurane nose cone on the rat 
SECTION: Blood Collection via Cardiac Puncture (NY, TN)
11.3. Spray fur with 70 % (v/v) EtOH 

SECTION: Blood Collection via Cardiac Puncture (NY, TN)
11.4. 
Using a forceps, grab a section of skin right above the abdominal cavity, and pull itup towards you while making a “Y” incision in the rat. Remove extra skin to expose the peritoneal cavity
  

SECTION: Blood Collection via Cardiac Puncture (NY, TN)
12. Blood Collection
SECTION: Blood Collection via Cardiac Puncture (NY, TN)
12.1. Open up a new needle and 5 mL syringe. 

SECTION: Blood Collection via Cardiac Puncture (NY, TN)
12.2. Holding sternum with forceps,cut through the diaphragm with scissors. 
SECTION: Blood Collection via Cardiac Puncture (NY, TN)
12.3. Insert the needle into the tip of the heart and draw up blood. Move the needle around to find a good position. 
SECTION: Blood Collection via Cardiac Puncture (NY, TN)
12.4. Inject blood into the serum separation tube. 
SECTION: Blood Collection via Cardiac Puncture (NY, TN)
12.5. Keep collecting until collect ~5mlCap the blood tube, invert 5 times, and place on ice until centrifugation.  

SECTION: Trunk Blood Collection (MI)
13. Trunk Blood Collection
SECTION: Trunk Blood Collection (MI)
13.1. Uncap a serum separation tube, put it on wet ice, and insert a funnel
SECTION: Trunk Blood Collection (MI)
13.2. Put rat into holder and into guillotine, decapitate animal over funnel
SECTION: Trunk Blood Collection (MI)
13.3. Hold animal over funnel, head side down. Collect all trunk blood coming out of the animal
SECTION: Trunk Blood Collection (MI)
13.4. Take out funnel and recap tube. Invert tube 5 times and put back on ice until tissue harvest is done. -


SECTION: Brain
14. Brain
SECTION: Brain
14.1. Cut off the head and hand over to the second person to perform a brain dissection. Record the time at the start of brain dissection. 
SECTION: Brain
14.2. If only one person is collecting the tissues and the brain, put the head on ice and dissect the brain after theliver tissue is frozen.
SECTION: Brain
14.3. Open the skull and remove the whole brain. Submerge it in the bucket of isopenten coooled in the container with dry ice (~-30 °C). When tissue is completely frozen (~30 s), gently wrap it in aluminum foil and transfer into the ½ oz jar and keep on dry ice.

SECTION: liver
15. Liver
SECTION: liver
15.1. Grab the top left liver lobe (left median lobe) with the forceps.
   

SECTION: liver
15.2. Pull the lobe up and cut it out being careful not to cut the large vein/artery that runs towards the back. 
SECTION: liver
15.3. Place a labeled ½ oz. jar on the scale and tare. 
SECTION: liver
15.4. Place the liver lobe in the jar.Weigh (to 1mg), record the weight, cap the container and place it on dry ice. 

SECTION: liver
15.5. Likewise, excise the right median liver lobe. 
SECTION: liver
15.6. Place a labeled ½ oz. jar on the scale and tare; Place liver lobe into vial, weigh (to 1mg), record weight, and place jar on dry ice
SECTION: Retroperitoneal Fat
16. Retroperitoneal Fat
SECTION: Retroperitoneal Fat
16.1. Move intestines/organs out of the way and locate the left kidney. 
SECTION: Retroperitoneal Fat
16.2. Grab the kidney with your finger and pull away the fat that is attached to it.
SECTION: Retroperitoneal Fat
16.3. Once kidney is disconnected, let go of the kidney and continue cutting/removing the fat from the cavity. 
  

SECTION: Retroperitoneal Fat
16.4. As you move closer to the testicles, you will see a faint “line” of fat.This is a landmark to determine where the fat pad ends.
SECTION: Retroperitoneal Fat
16.5. Cut along that line and remove the fat out of the rat  


SECTION: Retroperitoneal Fat
16.6. Place a labeled 1/2oz. jar on the scale and tare; 
SECTION: Retroperitoneal Fat
16.7. -dd the fat to the jar 
SECTION: Retroperitoneal Fat
16.8. Move the intestine/organs out of the way and locate the right kidney.
SECTION: Retroperitoneal Fat
16.9. Repeat the procedure above and remove the peritoneal fat on the right side.
SECTION: Retroperitoneal Fat
16.10. Place the right side fat in the same container that contains the left side fat, weigh in grams (to 1 mg). 
SECTION: Retroperitoneal Fat
16.11. Record the weight, cap the container and place it in dry ice
SECTION: Epididymis Fat (Male Rats)
17. Epididymis Fat (Male Rats) 
SECTION: Epididymis Fat (Male Rats)
17.1. Pull both testicles out of the body cavity.
SECTION: Epididymis Fat (Male Rats)
17.2. Take one testicle and look for the epididymis (curved area directly above the testicle)
SECTION: Epididymis Fat (Male Rats)
17.3. Hold the fat in one hand and carefully cut the fat away from the epididymis with scissors
  


SECTION: Epididymis Fat (Male Rats)
17.4. Place a labeled ½ oz. plastic jar on the scale and tare; add the fat to the container.
SECTION: Epididymis Fat (Male Rats)
17.5. Remove the epididymis fat from the other testicle.
SECTION: Epididymis Fat (Male Rats)
17.6. Add the new piece of epididymis fat in the same container with the first piece. 
SECTION: Epididymis Fat (Male Rats)
17.7. Record the weight in grams (to 1 mg), cap the container and place it on dry ice.
SECTION: Parametrial Fat (Female Rats)
18. Parametrial Fat (Female Rats) 
SECTION: Parametrial Fat (Female Rats)
18.1. Locate the two ovaries which are on top of the uterine horns.
SECTION: Parametrial Fat (Female Rats)
18.2.  While holding the ovary on the left side, cut the parametrial fat away from the kidney.The fat should only be attached to the uterine horn now.
 


SECTION: Parametrial Fat (Female Rats)
18.3. Continue to hold the ovary and trim the fat away from the uterine horn.Be sure to cut around the ovary.
  


SECTION: Parametrial Fat (Female Rats)
18.4. Place the storage container on the sale and tare it; 
SECTION: Parametrial Fat (Female Rats)
18.5. Place the left parametral fat in the container.
SECTION: Parametrial Fat (Female Rats)
18.6. Repeat the above steps for the right parametral fat.
SECTION: Parametrial Fat (Female Rats)
18.7. With both fat pads in the storage container, record the weight in grams (to 1 mg).
SECTION: Parametrial Fat (Female Rats)
18.8. Cap the container and place on dry ice.
SECTION: heart
19. Heart
SECTION: heart
19.1. Cut though the diaphragm with scissors (if no cardiac puncture).
SECTION: heart
19.2. Carefully grab the heart with forceps.
SECTION: heart
19.3. Cut the heart off by holding the heart with forceps, and cutting the aorta.
SECTION: heart
19.4.  Blot the heart on bench paper/gauze to get rid of extra blood.
 


SECTION: heart
19.5. Place a labeled ½ oz. plastic jar on the scale and tare. 
SECTION: heart
19.6. Place the heart in the jar, weigh in grams (to 1mg), record the weight.
SECTION: heart
19.7. Cap the container and place it on dry ice
SECTION: Left Kindey
20. Left Kidney 
SECTION: Left Kindey
20.1. Move intestines/organs to locate the left kidney.
SECTION: Left Kindey
20.2. Grab kidney with forceps.
SECTION: Left Kindey
20.3. Pull up towards yourself and cut the renal artery, vein, and urethra away from the kidney. 
SECTION: Left Kindey
20.4.  Remove any extra fat that may be attached to the kidney 


SECTION: Left Kindey
20.5. Place a labeled ½ oz. jar on the scale and tare.
SECTION: Left Kindey
20.6. Place the kidney in the jar. 
SECTION: Left Kindey
20.7. Weigh in grams, record the weight, cap the container and place on dry ice.
SECTION: Right Kidney
21. Right Kidney 
SECTION: Right Kidney
21.1. Dissect out the right kidney the same way the left kidney was dissected out.
SECTION: Right Kidney
21.2. Place a covered and labeled container with 10 % (v/v)ZBF Zinc formalin on the scale and tare (contains come pre-filled).

SECTION: Right Kidney
21.3. Remove the cover of the container, add the kidney to the container, cap the container, weigh it, and record the weight in grams.
SECTION: Right Kidney
21.4. Dissect out the right kidney the same way the left kidney was dissected out.
SECTION: Right Kidney
21.5. Place a covered and labeled container with 10% ZBF Zinc formalin on the scale and tare (containes come pre-filled).
SECTION: Right Kidney
21.6. Remove the cover of the container, add the kidney to the container, cap the container, weigh it, and record the weight in grams.
SECTION: Cecum
22. Cecum
SECTION: Cecum
22.1. Identify cecum in abdominal cavity. The cecum is found where the small and large intestines meet. It appears as a “dead end” structure.
SECTION: Cecum
22.2. Remove cecum by cutting immediately at the entrance of the small intestine to caecum and immediately below the exit of the caecum to large intestine.
SECTION: Cecum
22.3. Place isolated caecum and its contents into a sterile collection 50 ml tube.
SECTION: Cecum
22.4. -Place container on dry ice and store at -80 °C for.

SECTION: Cecum
22.5. This procedure should be performed as rapidly as possible to ensure integrity of the cecal contents.
  

SECTION: Right soleus
23. Right soleus
SECTION: Right soleus
23.1. Grab the rat’s right foot.
SECTION: Right soleus
23.2. Carefully cut a circle around the ankle being extra careful to only cut the skin and not any tendons underneath, using scissors.
   



SECTION: Right soleus
23.3. Remove the skin of the leg by grabbing a piece of the skin near the circle you just cut with forceps and pulling up towards the knee.

  

SECTION: Right soleus
23.4. Take a thin tipped forceps and poke a hole in the space between the Achilles tendon and connecting tissue. 
SECTION: Right soleus
23.5. Look at the rat’s lower leg; you’ll see a white line running the length of the gastrocnemius muscle.
SECTION: Right soleus
23.6. Place thin tipped forceps in the hole you just made by the Achilles tendon, and remove the outer covering of the gastrocnemius by pulling up along the white line.  


SECTION: Right soleus
23.7. Directly below that white line is the soleus. It appears as a brighter red color than the gastrocnemius and has tendons that attach to the area of the Achilles tendon and to the knee. 
SECTION: Right soleus
23.8.  Use the thin tipped forceps and pull forward the soleus by using the forceps as a spoon
 


SECTION: Right soleus
23.9. Find the end of the soleus tendon that attaches near the Achilles tendon. 
SECTION: Right soleus
23.10. Place a hemostat on the tendon. Make sure it’s on the tendon and not the muscle! 
SECTION: Right soleus
23.11.  Using small scissors, cut the tendon to release the muscle from the leg.
 


SECTION: Right soleus
23.12. Leaving the hemostat connected, pull the hemostat up towards you to reveal the location of where the soleus is connected by the knee. 
SECTION: Right soleus
23.13. Cut the tendon connected to the soleus to completely remove the soleus from the leg.
SECTION: Right soleus
23.14.  Remove the hemostat
 


SECTION: Right soleus
23.15. Place the soleus in a labeled ½ oz. jar and place on dry ice, there is no need to weigh it.
SECTION: Spleen
24. Spleen
SECTION: Spleen
24.1. Return to the spleen. 
SECTION: Spleen
24.2. Remove spleen and place into 2 ml tube containing 0.5 ml saline.
  

SECTION: Spleen
24.3. Locate the RFID tag in the rat (it may have moved from its original subcutaneous location). 
SECTION: Spleen
24.4. Place the RFID tag into the ½ oz. jar along with the spleen. 
SECTION: Spleen
24.5.  Place tube on dry ice.
 

SECTION: Baculum
25. Baculum
SECTION: Baculum
25.1. With your thumbs, push into the abdomen on either side of the penisuntil the penis pokes out, grab the penis with forceps, pull it out some more, cut off with scissors at the base.
SECTION: Baculum
25.2. Put directly in the labeled2 mL tube and freeze.

SECTION: Right Eye
26. Right eye 
SECTION: Right Eye
26.1. Use scissors to make incisions at lateral and medial canthi at the right eye.
  


SECTION: Right Eye
26.2. Insert forceps with curved tips (Dumont #5, angled) under eye being careful not to puncture eye.
SECTION: Right Eye
26.3. Gently pull up on tightly closed forceps to stretch extraocular muscles. Be careful not to pinch or crush eye.
SECTION: Right Eye
26.4. Gently pull until eye is pulled from socket.  


SECTION: Right Eye
26.5. Eye should be spherical and have optic nerve attached. 
  

SECTION: Right Eye
26.6. If dissection is unsuccessful, dissect the left eye, make a note in the file.
SECTION: Right Eye
26.7. Put 1 mL of Karnovsky’s fix in labeled 7 mLvial. 

SECTION: Right Eye
26.8. Put eye into the vial. 
SECTION: Right Eye
26.9. Store at 4 °C. 

SECTION: Right Eye
26.10.  Ship on wet ice.
 
SECTION: hindlimb
27. Hindlimb
SECTION: hindlimb
27.1. -Put rat in the prone position, head facing away from you, 
SECTION: hindlimb
27.2. Retract left leg (left leg works better for the right handed person) away from the body and approach with the scissors from the back of the thigh. Cut through the skin and soft tissues (muscle) as close to the body as possible. Aim to locate the femur, but don’t cut it. Instead, move the tip of the scissors along the femur in the direction of the hip joint ). You will be able to find the capsule of the hip joint if you go ~1/8 of an inch from the proximal tip of the shaft of the femur towards the tail (if you think of your forearm as a shaft of the femur, the neck of the femur sticks out as a thumb of your left hand when its palm faces down). It will be easier to spot the joint if you keep moving that hip with your left hand. 
SECTION: hindlimb
27.3. Cut through the capsule of the joint with the tips of the scissors and that will release the head of the femur. 
SECTION: hindlimb
27.4. Cut through the soft tissues and the skin in the front of the thigh. 
SECTION: hindlimb
27.5. Put the entire leg into barcoded 50 mL tube and put it on dry ice.

SECTION: hindlimb
27.6. Transfer it to a-80 °C freezer.

SECTION: Rest of carcass
28. Rest of carcass 
SECTION: Rest of carcass
28.1. Remove the whole digestive tract and any other remaining internal organs
SECTION: Rest of carcass
28.2. Remove remainder of tail (3/4 inch of tail was collected earlier) and discard.
SECTION: Rest of carcass
28.3. Remove remainder of head and discard (if head has not been removed previously).
SECTION: Rest of carcass
28.4. Affix a barcode label inside of a Ziploc bag (sandwich or quart size, as available) and affix a second label on the outside. 
SECTION: Rest of carcass
28.5. Place carcass into Ziploc bag and freeze at -20 °C. 

SECTION: blood tubes
29. bloodtubes 
SECTION: blood tubes
29.1. Centrifuge blood whithn 1 hour after collection. If dissection takes 15 min, blood can be processed in butches of 4.
SECTION: blood tubes
29.2. Put the serum separation tube intoa 50 ml conical tube. Put into4 degree centrifuge and spin at 3,000 rpm for 18 minutes
SECTION: blood tubes
29.3. This step can be adjusted: depending on centrifuge you can spin the original SST tube; you can spin longer at lower speed.
SECTION: blood tubes
29.4. Once finished spinning, the beige colored “plug” in the blood tube will separated out the serum from the red blood cells.If the plug hasn’t moved yet, spin again for a few minutes.
SECTION: blood tubes
29.5. Place microcentrifuge tubes in labeled boxes and freeze at -80 °C.