CREB3L2-ATF4 heterodimers were visualized using Duolink In Situ Brightfield Detection reagents (DUO92012, MilliporeSigma). CREB3L2 and ATF4 PLA probes were prepared as described for the detection of CREB3L2-ATF4 heterodimers in 5xFAD mice. Per manufacturer’s instructions, the PLA Probe Diluent included in the Probemaker Kit was used in substitution of the PLA Antibody Diluent in the PLA protocol. Before deparaffinization with xylene, slides were placed in a 60°C oven for 1 hour; we proceeded by rehydrating slides using a graded ethanol series (100% > 95% > 70% > 50% > water) plus two 10-min PBS-T washes. Epitope unmasking was done for 20 min in steaming tris-EDTA buffer [10 mM tris base, 1 mM EDTA, and 0.05% Tween 20 (pH 9.0)], followed by three 5-min PBS-T rinses.
We quenched endogenous peroxidases slides with 1% hydrogen peroxide for 30 min before blocking. Costaining of neurofilament (1:400; heavy chain subunit; #N0142, MilliporeSigma) was performed afterward using the Vector Blue Alkaline Phosphatase Substrate Kit (SK-5300, Vector Laboratories). To increase detection sensitivity, we additionally used the Vectastain ABC-AP system (AK-5002, Vector Laboratories) before signal development. Last, sections were dehydrated in a graded ethanol series (50% > 70% > 95% > 100%), cleared with Histo-Clear (64110-01, Electron Microscopy Sciences), mounted in VectaMount (H-5000, Vector Laboratories), and air-dried for 24 hours before proceeding with imaging. Human dorsolateral prefrontal cortex specimens (Brodmann area 8/9; table S2) were manually counted by an experimenter “blind” to the underlying diagnosis. Technical controls: PLA Probe Rabbit IgG Isotype Control MINUS (DUO87004, MilliporeSigma) and CREB3L2 blocking peptide (APrEST73339, Atlas Antibodies). For each case, CREB3L2-ATF4 measurements were interspersed between five randomly selected tissue subregions; specifically, 10 neurons within layers III to V were analyzed in each subregion, for a total of 50 independent measurements per brain.
We quenched endogenous peroxidases slides with 1% hydrogen peroxide for 30 min before blocking. Costaining of neurofilament (1:400; heavy chain subunit; #N0142, MilliporeSigma) was performed afterward using the Vector Blue Alkaline Phosphatase Substrate Kit (SK-5300, Vector Laboratories). To increase detection sensitivity, we additionally used the Vectastain ABC-AP system (AK-5002, Vector Laboratories) before signal development. Last, sections were dehydrated in a graded ethanol series (50% > 70% > 95% > 100%), cleared with Histo-Clear (64110-01, Electron Microscopy Sciences), mounted in VectaMount (H-5000, Vector Laboratories), and air-dried for 24 hours before proceeding with imaging. Human dorsolateral prefrontal cortex specimens (Brodmann area 8/9; table S2) were manually counted by an experimenter “blind” to the underlying diagnosis. Technical controls: PLA Probe Rabbit IgG Isotype Control MINUS (DUO87004, MilliporeSigma) and CREB3L2 blocking peptide (APrEST73339, Atlas Antibodies). For each case, CREB3L2-ATF4 measurements were interspersed between five randomly selected tissue subregions; specifically, 10 neurons within layers III to V were analyzed in each subregion, for a total of 50 independent measurements per brain.
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