Universal lsab kit hrp

For immunohistochemical staining, the TMAs were first deparaffinized and then rehydrated in a series of ethanol baths (100%, 90%, 75% and 50%). Antigen retrieval was achieved by citrate buffer (pH 6.0) in a pressure-cooker (15 psi) for 30 min. Immunostaining for HSET (1:1000 dilution) was performed using a rabbit polyclonal antibody which was a generous gift from Claire Walczak (Indiana University). Enzymatic antibody detection was performed using Universal LSAB + kit/HRP (DAKO, CA, USA). HSET staining was scored for both the nuclear and cytoplasmic localization as an intensity and frequency score by an experienced pathologist. A relative intensity score was represented as 0 = none, 1 = low, 2 = moderate, or 3 = high and frequency score was represented as the percentage of cell nuclei or cytoplasms demonstrating HSET positivity.

Placental tissue (n = 8 for IVP; n = 9 for CTR) collected on day 20 of gestation were fixed in 4% paraformaldehyde overnight, dehydrated with increasing ethanol grandient (5’ each step), cleared in xylene mixture (5’ minute each step) and finally paraplast embedded. 5 μm sections were dewaxed and rehydrated. Then, one part was used for hematoxylin eosin staining and other for immunohistochemistry analysis. First group of the sections were stained with Hematoxylin Solution (Bio-Optica, Milan, Italy), differentiated in acid alcohol solution, counterstained with Eosin Solution (Bio-Optica, Milan, Italy) and then dehydrated in 100% ethanol and cleared in xylene mixture. For immunohistochemistry, tissue sections were incubated overnight at room temperature with 4 μg/ml of primary rabbit polyclonal antibody against LC3 (ab58610; Abcam, Cambridge, UK). Negative control:tissue sections were overnight incubated without primary antibody. Antibody binding was visualized using Universal LSAB^™ Kit/HRP and Dako Liquid DAB Substrate Chromogen (Dako, Glostrup, Denmark). Images were captured using a Nikon Eclipse E600 light microscope (Melville, NY, USA).