Vector immpact novared peroxidase substrate kit

Paraffin-embedded spinal cord and motor cortex tissue sections from 5 sporadic ALS cases, 2 disease controls (DCs) and 2 non-neurologic diseased controls (NNDCs) were obtained from the Barrow Neurological Institute and Target ALS post-mortem tissue bank cores (subject demographics listed in Table 1). Participants of the post-mortem tissue bank cores provided IRB approved informed consent for the collection of post-mortem tissues. Immunohistochemistry (IHC) was performed as previously described^{13 (link), 34} . Tissue sections were de-paraffinized and rehydrated, and antigen retrieval was performed using the Antigen Retrieval Citra Solution (pH 6) (BioGenex). Tissues were blocked using Super Block supplemented with Avidin (Vector Labs) and incubated with rabbit anti-SNRNP200 antibody (Sigma) diluted in Super Block supplemented with biotin (Vector Labs) overnight. An anti-rabbit biotinylated IgG secondary antibody (Vector Labs) was subsequently added, and immunostaining was visualized using the Vectastain Elite ABC reagent (Vector Labs) and Vector ImmPACT NovaRED peroxidase substrate kit (Vector Labs). Slides were counterstained with hematoxylin (Sigma). Images were acquired using an Olympus BX40 microscope and a minimum of 11 motor neurons were quantified for each case using Image J³⁴ . Details on antibodies used for IHC are listed in Table S1.

Paraffin-embedded post-mortem frontal cortex tissue sections were used for this study. All sections were deparaffinized, rehydrated and antigen retrieval performed using Target Antigen Retrieval Solution, pH 9.0 (DAKO) for 20 min in a steamer. After cooling to room temperature, non-specific binding sites were blocked using Super Block (Scytek), supplemented with Avidin (Vector Labs). Primary antibodies used for immunohistochemistry were incubated overnight in Super Block with Biotin. Slides were then washed and incubated for 1 h in the appropriate biotinylated IgG secondary antibodies (1:200; Vector Labs) in Super Block. Slides were washed in PBS and immunostaining visualized using the Vectastain Elite ABC reagent (Vector Labs) and Vector Immpact NovaRED peroxidase substrate kit (Vector Labs). Slides were counterstained with hematoxylin (Sigma Aldrich) and pictures were captured using an OLYMPUS BX40 microscope equipped with a SebaCam camera.