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Renal transporters (cotransporters, channels, and claudins) mediate homeostasis of fluids and electrolytes and are targets of hormonal and therapeutic regulators. Assessing renal transporter abundance with antibody probes by immunoblotting is an essential tool for mechanistic studies. Although journals require authors to demonstrate antibody specificity, there are no consensus guidelines for kidney sample preparation leading to lab-to-lab variability in immunoblot results. In this study, we determined the impact of sample preparation, specifically freeze-thawed (Frozen) versus freshly processed (Fresh) kidneys (female and male rats and mice) on immunoblot signal detection of 15 renal transporters and the impact of protease inhibitors during homogenization. In female Sprague-Dawley rat kidneys homogenized with aprotinin, Na2EDTA, PMSF, and phosphatase inhibitors, immunodetection signals were ∼50% lower in Frozen versus Fresh samples for most transporters. Inclusion of additional inhibitors (Roche cOmplete Protease Inhibitor, "+") only partially increased transporter immunoblot signals to near Fresh levels. In male Sprague-Dawley rats, immunoblot signal density was lower in Frozen+ versus Fresh+ despite additional inhibitors. In C57BL/6 male mice, immunoblot signals from proximal tubule transporters were lower in Frozen versus Fresh by ∼25–50% and greater in Frozen+. In contrast, immunodetection signal was equivalent in female Frozen+ versus female Fresh+ for claudin 2, villin, AQP1, NKCC2, NCC, ENaCβ, ENaCɣ, claudin 7, AQP2, NKAα1, and NKAβ1. Thus, kidney sample preparation variables, including freeze-thaw and protease inhibition, have substantial transporter-specific effects on quantification of renal transporter abundance by immunoblot. These findings underscore the critical importance of assessing and reporting the impact of sample preparation protocols on transporter recovery to ensure robust rigor and reproducibility. NEW & NOTEWORTHY: Freeze-thawing kidneys before homogenization is widely accepted in renal research. This study demonstrates that if kidneys are freeze-thawed just once before homogenization, immunoblot signals are reduced in a transporter-specific manner in rats and mice dependent on sex and that immunoblot signals can be partially recovered by adding additional protease inhibitors. These findings underscore the critical importance of assessing the impact of sample preparation, including freeze-thaw versus fresh, to ensure robust rigor and reproducibility. [ABSTRACT FROM AUTHOR]