Immpress excel amplified polymer staining kit

PDOs were pooled from 3 to 5 wells and incubated for 1.5 h on ice in cell recovery buffer. PDOs were washed with PBS, spun down at 500 rpm for 3 min, and then fixed in 4% PFA for 30 min. Fixed PDOs were stained for 5 min with hematoxylin, washed with water and resuspended in warm Histogel (Epredia HG400001). The Histogel and PDO mix was moved to an insert with 200 μl cooled Histogel and placed on ice for 15 min. Solidified Histogel containing the PDOs was placed in a cassette, fixed overnight in 10% formalin and embedded into paraffin. After deparaffinization, the sections were treated with low pH citrate buffer in a microwave at full power for 2 min and then 10 min at low power. Immunohistochemistry for estrogen receptor (Abcam #ab16666) was performed using the ImmPRESS Excel Amplified Polymer Staining kit (Vector laboratories #MP-7601) according to the manufacturer’s description. The sections were imaged using an Olympus brightfield microscope and scored by a trained pathologist for nuclear features and ER staining.

Specimens were fixed in 4% paraformaldehyde at indicated time point for 24 h overnight at 4C before being dehydrated and paraffin embedded. Forelimbs were sectioned at 7 μm thickness. For Safranin O/Fast Green staining, sections were deparaffinized and rehydrated before being stained in Weigert’s Iron Hematoxylin (Sigma) and 0.02% aqueous Fast Green (Sigma) followed by a rinse in 1% acetic acid and 0.1% aqueous Safranin-O (Sigma). For immunohistochemistry, ImmPRESS Excel amplified polymer staining kit (Vector Labs) was used [60 ]. Primary antibodies used for immunohistochemistry were rabbit anti-Osx/Sp7 (1:500) (Abcam, Ab22552), rabbit anti-Runx2 (1:500) (Santa Cruz, Sc-10,758 M-70), and rabbit anti-Gli1 (Santa Cruz, Sc-20,687 H-300) (1:200). For immunocytochemistry, mesenchymal progenitors were plated on chamber slides and allowed to adhere for 24 h. Cells were then fixed with 4% paraformaldehyde and stained with mouse anti-acetylated alpha tubulin (1:200) (R&D systems T7451 6-11B-1) overnight at 4C followed by chicken anti-mouse Alexafluor 647 (1:200) (Invitrogen) and DAPI counterstain.