Introduction

Explore detailed protocols on rat tissue preparation for liver, lung, and blood. The rat liver preparation includes steps for aseptic removal, flash-freezing, and homogenization. The rat lung perfusion protocol outlines methods for effective perfusion, including pulmonary artery cannulation. Learn rat blood collection through cardiac puncture using both open and closed chest methods, helping ensure sample purity and consistency.

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Rat liver preparation

Protocol tips

Add protease inhibitor directly to PBS. We recommend using Halt Protease Inhibitor Cocktail, reconstituted to 1X (10 μL of 100X Halt Protease Inhibitor Cocktail per mL of buffer). The stability of protease inhibitor-supplemented buffer is 24 hours at 4°C.

Materials

Procedure

  1. Remove liver aseptically.
  1. Flash-freeze liver in foil packets using liquid nitrogen. Store at –80°C.
  1. Crush liver sample on dry ice and store in a prechilled 5 mL culture tube.
  1. Add about 100 μg tissue (enough to almost cover round portion of a 5 mL culture tube) to a new chilled tube.
  1. Add 1 mL PBS with 10 μL of 1X Halt Protease Inhibitor Cocktail. Chill this solution using wet ice.
  1. Homogenize at low speed for ~20 seconds. Make sure to keep cool and keep samples on wet ice.
  1. Transfer samples into clean 1.7 mL microcentrifuge tubes.
  1. Centrifuge at 14,000 x g for 15 minutes at 4°C.
  1. Remove and aliquot supernatant. We recommend making several 50 μL aliquots.
  1. Before running ELISA, dilute the protein sample at a ratio of 1:100. Determine protein concentration using protein assay of your choice such as BCA protein assays or ready-to-use Quant-iT Reagents and Kits for 20-2,000 Samples, such as the Quant-iT Protein Assay Kit.
  1. Dilute the samples to equal concentrations and then run an ELISA. Refer to General ELISA Protocols or follow the instructions provided with the ELISA kit.


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Rat lung perfusion

Materials

  • Dissecting microscope or eye magnifier
  • Phosphate-buffered saline (PBS, pH 7.2)
  • Cotton swab
  • Surgical scissors or scalpel
  • 10 mL syringes (quantity 2) with a stopcock and a small gauge needle (25 or 27 ga) attached

Procedure

  1. Rat should be fully anesthetized (e.g., unresponsive to toe pinch).
  1. Euthanize the rat by exsanguination. This can be done by cutting the abdominal aorta. Avoid cardiac puncture, as the puncture site will leak during the perfusion procedure.
  1. Open the chest widely, taking care not to puncture the lungs. Cut through the entire sternum at the midline; lifting the sternum with forceps and keeping the scissor tips horizontal will lessen the chance of inadvertent lung puncture. Next, make lateral incisions in the chest wall in both directions from the midline incision. Retract one side of the chest wall laterally, bluntly dissect the pleura to free up the lung, gently push the lung away from the chest wall with a cotton swab, and cut the chest wall vertically along the spinal processes. Repeat for the other half of the chest wall. Be careful not to puncture the lung with the sharp edges of the cut ribs.
  1. Place vascular clamps or tie ligatures on both the superior and inferior vena cava to prevent backflow of perfusion buffer into the systemic venous system. It helps to use magnification (dissecting microscope or eye magnifiers) for the remaining steps.
  1. With a scalpel or small scissors, make an incision in the LEFT ventricle of the heart, making certain that the heart chamber is open to the outside.
  1. Prepare a 10 mL syringe filled with PBS, with a stopcock and a small gauge needle (25 or 27 ga) attached. Keep a second syringe filled with PBS on hand in case it is needed. Insert the needle into the RIGHT ventricle, and gently push the PBS into the pulmonary circulation. You should see the right heart and pulmonary artery swell a little bit, and fluid should immediately begin to flow from the left ventricle incision. Continue to push PBS through until the lungs are white and the fluid coming out of the left ventricle is completely clear- this usually takes 10–15 mL of buffer. The small needle will prevent you from pushing in the buffer too rapidly, and also will have fewer problems with leaks than with larger needles.
  1. Another effective option for perfusion, although more time-consuming, is to cannulate the pulmonary artery. This can be done by introducing a plastic catheter through the right heart and into the pulmonary artery, and ligating the pulmonary artery to hold the catheter in place. With this method it is not necessary to clamp the vena cava. Buffer may be pushed into the catheter with a syringe, taking care not to create excessive pressure in the pulmonary system, or with a gravity feed of buffer not more than about 10 cm above heart level.


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Rat blood collection

Materials

  • Hemostat
  • Thumb forceps
  • Surgical scissors or scalpel
  • Syringe (3–10 mL) with needle (20 ga) attached

Procedure

Rat should be fully anesthetized (e.g., unresponsive to toe pinch).

Open chest method

  1. Prepare a syringe (3–10 mL) containing ~0.3 ml of air, fitted with a 20 gauge needle.
  1. Make an incision at the level of the xiphoid process and hold the xiphoid with a hemostat or thumb forceps.
  1. Beginning at the xiphoid, cut along the full length of the sternum just to the rat’s right of the midline, then make a lateral incision through the chest wall laterally to the rat’s left, just cranial to the diaphragm. Do this quickly, as there will be arterial bleeding from the chest wall incisions.
  1. Continue to hold the xiphoid, retract the chest wall flap laterally, and expose the heart with blunt dissection of the pleura and pericardium. If necessary, make a lateral incision to the right in the chest wall (there should be adequate exposure of the heart without the second lateral incision if the initial midline incision is slightly to the right of the sternum).
  1. Insert the needleinto the right ventricle and apply gentle negative pressure to the syringe to fill the syringe with blood. Excessive negative pressure will collapse the chamber. The right atrium also works well for drawing blood, but the left side of the heart is more difficult.

Closed chest method

  1. Prepare a syringe (3–10 mL) containing ~0.3 ml of air, fitted with a 20 gauge needle.
  1. Make an incision in the skin and abdominal wall, caudal to the sternum.
  1. Using a hemostat or thumb forceps applied to the xiphoid process, retract the sternum cranially until the apex of the heart is visible against the diaphragm.
  1. Insert the needle through the diaphragm and into the heart, applying gentle negative pressure once the heart has been punctured, and reposition the needle as necessary until blood flows into the syringe. Avoid excessive negative pressure, as it will collapse the heart chamber onto the needle tip.


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For Research Use Only. Not for use in diagnostic procedures.