The first crucial step for Core activities will be communication with Investigators through expert Core personnel on how to prepare kidney tissue and how to harvest, section and examine tissues at time of sacrifice. These prepared materials can then be transported to the Core facility for appropriate processing.
A) Obtaining tissue
Principle: Drawing blood prior to tissue harvest induces artifactual changes, particularly in kidney tissue. It is therefore recommended to follow the procedure outlined below. In the anesthesized animal (i.e. rat/mouse):
1) Perform laparotomy followed by visualization of kidneys.
2) Prior to harvesting organs and obtaining blood samples, place a hemostat at vascular pole of kidney; be sure to include both artery and vein when clamping.
3) Surgically remove the kidney(s) while hemostat remains clamped over the vessels. This maintains blood flow in aorta and prevents hemorrhage from renal artery.
4) Process and section the kidney as described below or as indicated by protocol for a specific experimental procedure.
5) Blood may now be obtained from either aorta or by cardiac puncture, followed by harvest of other organs as indicated.
B) Allocation of tissue for morphologic and molecular studies
Principle: Allocate tissue samples in both Carnoy’s fixative and 4% paraformaldehyde for immunostaining/immunohistochemistry (IHC) and in situ hybridization studies.
A portion of the tissue sample should be frozen for isolation of mRNA and another portion should be frozen separately for ECM quantification and/or protein analysis.
Special studies may require specific protocols of tissue preservation and should be discussed with Core personnel. Contact information can be found on the MKPDC web page.
Light microscopy: Place a 2 mm thick coronal section from the mid-portion of the kidney in 4% paraformaldehyde/PBS.
Immunohistochemistry/in situ hybridization: Place a similar portion of kidney tissue in Carnoy’s fixative.
mRNA or Western blot: The larger of the two remaining poles of the kidney should be frozen for mRNA isolation. The other pole may be frozen ECM quantification and/or protein studies.
EM studies: Cut small cubes (1-2 mm) from the area of interest and place them in 2% glutaraldehyde fixative.
Special considerations for tissue allocation exist for specific models:
UUO (unilateral ureteral obstruction): harvest both kidneys, designate separately and allocate each individual kidney as described above (the contralateral kidney serves as a control).
Remnant kidney model: At time of 5/6 nephrectomy, process the removed kidney (baseline kidney) as described above and retain it for paired studies with remnant kidney. At time of harvest of remnant kidney obtain 1-2 mm piece from mid portion for LM/IHC studies. Freeze upper pole for isolation of mRNA.
Note: Do not allocate necrotic tissue for studies.
For frozen sections, portions of the kidneys should be directly embedded in OCT and rapidly frozen on dry ice
A further major function of the Core will be processing tissue samples for various stains, including hematoxylin and eosin (H&E), periodic acid Schiff (PAS), Jones’ silver stain and Masson trichrome stain (see example below).