Envision flex hrp kit

Splenic tissue was fixed in 10% neutral buffered formalin and then embedded in paraffin. To remove paraffin and rehydrate splenic sections a PT Link device (DAKO, Stockport UK) was used according to the manufacturer's instructions. Immunohistochemical staining was performed using a DAKO Auto-stainer automated slide processing system and the EnVision™ FLEX/HRP kit (DAKO, K8012). The primary antibodies used were PKCβII (C-18) and HA (C29F4). Hematoxylin and eosin (H&E) staining of splenic sections was carried out according to standard protocols.

To determine the pathogenesis of alpha 2u-globulin nephropathy induced by STZ injection, as relevant to water absorption and glomerular filtrate function via AQP, immunohistochemical (IHC) and immunofluorescence (IF) studies were conducted using EnVision FLEX/HRP kit (DAKO, Denmark) and VectaFluor Duet immunofluorescence double labelling kit, DyLight 488 Anti-rabbit (green)/DyLight 594 Anti-mouse (red) (VECTOR, USA), respectively. The sections were deparaffinised in xylene, hydrated in a series of graded ethanol and heat-retrieved to enhance the antigenicity in citrate buffer, pH 6.0. Polyclonal rabbit anti-AQP-1, − 2, − 4 and − 5 (Millipore, USA) antibodies were incubated on the tissues. Appropriate secondary antibodies matching their conjugate and visualisation system from the kit were applied to the sections. The nuclei were counterstained by either haematoxylin or VECTASHIELD Antifade mounting medium with DAPI (VECTOR, USA) for IHC and IF, respectively. Immunolocalisation was measured using the H-score as mentioned above. In addition, the area of expression as a percentage was determined using an image analysis programme (ImageJ, version 1.51 J8, NIH). Briefly, five images of labelled areas were captured and transformed to binary images. Immunolocalisation was defined by the threshold mode and determined as an area fraction (%).

Immunostaining for cleaved caspase-3 (Asp175) was performed using rabbit plolyclonal antibodies against amino-terminal residues adjacent to (Asp175) in human caspase 3. Sections measuring 2 μm were cut from each paraffin block and were put to dry at 37°C during 12 h. After deparaffinization and rehydration, all sections were pretreated at pH 6 with Flex TRS Low (PTLink DAKO, Glostrup, Denmark) during 20 min. Endogenous peroxidase was blocked in 1% hydrogen peroxide for 5 min (DAKO, Glostrup, Denmark) at room temperature. After rinsing with phosphate buffered saline, the sections were incubated with cleaved caspase-3 (Asp175) antibody (#9661, Cell Signaling) for 20 min at room temperature. Then sections were incubated with an appropriate secondary antibody from the Envision flex/HRP kit (Dako, Glostrup, Denmark) for 20 min, at room temperature. Next, slides were incubated in PBS, for 20 min, at room temperature, and then peroxidase activity was detected by diaminobenzidinetetrahydrochloride for 8 min and used for visualization and haematoxylin (Dako, Glostrup, Denmark) during 6 min for nuclear counterstaining.