CLINICS 2012;67(3):255-259 Defrosted isolated total RNA is preserved Olivera IB et al. Figure 3A - The A260/A280 ratios of the total RNA in unthawed (control) and thawed isolated total RNA samples. Figure 3B - RNA concentration in unthawed (control) and thawed isolated total RNA samples. The proteins were transferred onto a 0.20 mm pure nitrocel- lulose membrane (Bio-Rad Laboratories Inc, Hercules, CA, USA, http://www.bio-rad.com) for 1 h at 300 mA constant current using a semidry transfer cell (Bio-Rad Laboratories Inc, Hercules, CA, USA, http://www.bio-rad.com). The gel was stained with Coomassie blue to determine whether the proteins had been transferred to the membrane. To ensure an adequate protein transfer, the membranes were stained with Ponceau S. The blots were blocked overnight at 4˚C with 5% nonfat dry milk in Tris-buffered saline (1 mol/L Tris-HCl [pH 7.5] and 3 mol/L NaCl). The membrane was washed with Tris-buffered saline plus Tween (TBST) to remove the Ponceau S and then incubated overnight at 4˚C with 1:5000 anti-beta-actin primary antibody (Dako Denmark A/S, Glostrup, Denmark, http://www.dako. com). After washing the blots in TBST, they were incubated with a secondary anti-mouse IgG-horseradish peroxidase-conjugated antibody (Dako Denmark A/S, Glostrup, Denmark, http:// www.dako.com) at 1:5000 for 2 hours. The blots were then washed again in TBST. The proteins were visualized using a chemiluminescent agent (GE Healthcare, Piscatway, Uppsla, USA, http://www.gehealthcare.com) and autoradiography with pre-flashed Kodak film (Eastman Kodak Company, Rochester, NY, USA, http://www.kodak.com). The intensity of the individual bands was quantified by optical densitometry using Scion Image for Windows (Scion Corporation, Maryland, USA, http://scion-corporation.soft- ware.informer.com). A pre-stained protein marker (Bio-Rad Laboratories Inc, Hercules, CA, USA, http://www.bio-rad. com) was used as the molecular weight standard. Histology The thawed tissues [pancreas (n = 3), white (n = 4) and brown (n = 3) adipose tissue, kidney (n = 3), liver (n = 3), and heart (n = 4)] were fixed in 10% buffered formalin and embedded in paraffin blocks. The morphological analysis was performed as described (8). Briefly, 5-mm-thick sections were cut and stained with Masson’s trichrome and periodic acid Schiff for structural analyses. The tissue sections were examined using a magnification of 200X (Nikon Instruments Inc, Melville, NY, USA, http://www.nikoninstruments.com). Statistical procedures The data are expressed as the mean ¡ SEM. The normality of the distributions was tested before performing Student’s t Figure 4A - Integrity of the thawed and unthawed isolated total RNA samples. 1 = ladder; 2 and 3 = unthawed kidney (control); 4 to 18 = thawed isolated total kidney RNA. Figure 4B - Renin mRNA level in thawed isolated total kidney RNA. Figure 4C - b-Actin mRNA level in thawed isolated total kidney RNA. 257