Tissue Preparation and Immunohistochemistry

Each brain was fixed in formalin for at least 2 weeks, cut into 2- to 3-cm-thick blocks and placed in increasing gradients of sucrose (10%–40%) for cryoprotection. Small blocks from various cortical and subcortical regions were embedded in paraffin, cut at a thickness of 5 µm, and used for pathological diagnosis and for qualitative comparison of TDP-43 inclusions and pre-inclusions between PPA and controls participants. For pathological diagnosis, standard staining protocols, including staining for phosphorylated tau, amyloid-β, α-synuclein, and TDP-43, were employed. All brains were examined for gross and microscopic pathology. Brains of cognitively normal participants were free of pathology except for age-appropriate changes, including low densities and restricted distribution of plaques and tangles. Brains of PPA cases were diagnosed with FTLD-TDP type A pathology or type B pathology (Table 1). FTLD-TDP type A is characterized by many NCIs and short DNs, predominantly in layer 2, while type B consists of moderate NCIs and few DNs throughout all cell layers (17). In addition to the primary TDP proteinopathy, some of the PPA brains contained secondary pathologies. These included ALS-type motor neuron loss (participants 3 and 4, Table 1), mild vascular changes (participants 1 and 3), cerebellar vermis and mammillary body atrophy (participant 1), hippocampal sclerosis, and brainstem Lewy bodies (participant 2).

For determination of pre-inclusion types, distribution and quantitation, whole hemisphere blocks from brains of PPA participants containing regions of interest (ROIs) were cut in the coronal plane at 40-µm thickness on a freezing microtome (Leica SM2010R, Nussloch, Germany). Every 1 in 24 sections was immunohistochemically stained using the avidin-biotin peroxidase (ABC) method, with an antibody against TDP-43 phosphorylated at serine residues 409/410 (pS409/410-2, rabbit polyclonal, 1:3000, CosmoBio, Carlsbad, CA) to visualize inclusions and pre-inclusions. All sections were counterstained with 0.05% Cresyl violet to aid in subcellular localization of the TDP-43 inclusions in neurons.