Eosin y solution

Rat kidneys were freshly fixed in 4% PFA, washed in RNase free water and transferred in 70% RNase free ethanol. Kidneys were bisected longitudinal before automated embedding in paraffin using a STP120 (Thermo Fisher Scientific). Each paraffin-embedded half was sectioned (10-μm sections) using a microtome HM340E (Thermo Fisher Scientific).
Histologic staining was performed on deparaffinized and hydrated serial sections of rat kidney. Hematoxylin and eosin (H&E) staining visualized cell nuclei (black, dark blue) and counterstains cytoplasm and connective tissue fibers (different shades of pink). In detail (also shown in Table 1), staining was started by deparaffinization with two steps of absolute xylene followed by rehydration steps with a descending ethanol row. Hematoxylin staining was done with Mayer’s hematoxylin solution (Carl Roth GmbH, T865.1) for 10 min followed by 10 min bluing in lukewarm running tap water. Counterstain was done with 1% Eosin Y solution (Carl Roth GmbH, 3137.2) for 2 min followed by a differentiation step in 70% ethanol for 30 s. Stained sections were mounted with Roti-Histokitt (Carl Roth GmbH, 6638.1). Stained sections were stored at room temperature until imaging analysis was performed.

Sections of Bouin-fixed testis were deparaffinized, hydrated, stained with Hemalum solution acid (Henricks and Mayer, 1965 (link)) and Eosin Y solution (Carl Roth), dehydrated and mounted with Entellan (Sigma-Aldrich/Merck). Tubule diameters were determined by measuring the horizontal and vertical diameters of at least 25 tubules per testis cross-section. The number of elongated spermatids per tubules for a minimum of five tubules per mouse was counted with the ImageJ cell counter. Images were white balanced using the ImageJ macro ‘White balance correction_1.0’ originally written by Vytas Bindokas (2006, University of Chicago, USA) and modified by Patrice Mascalchi (2014, University of Cambridge, UK).