2.4. Immunohistochemical staining

C57BL/6J and APP/PS1 mice that were implanted at ages of 2 months old were sacrificed and perfused according to University of Pittsburgh IACUC approved methods at 1 week (n= 6 per group) or 16 weeks (n = 7 per group) post-implantation, as described previously [7]. Briefly, mice were sedated using a cocktail mixture of xylazine (7 mg kg⁻¹) and ketamine (75 mg kg⁻¹). A toe-pinch test was performed to ensure proper level of anesthesia prior to beginning the procedure. For each mouse, 100 ml of warm phosphate buffered saline (PBS) was perfused transcardially (pump pressure between 80–100 mm Hg) followed by 100 ml of 4% paraformaldehyde (PFA). Mice were then decapitated with both skull and implant left intact for 24 h post-fixation at 4 °C in 4% PFA. This extended fixation period ensured the tissue surrounding the microelectrode remains minimally disturbed during implant removal. Brains were then carefully detached from the skull by removing excess bone and gently pushing down on the headcap while the tissue sample is inverted, allowing the probe to fall straight down out of the brain. Brain samples were then sequentially soaked in 15% and 30% sucrose in PBS at 4 °C for 24 h each. Once the tissue had reached sucrose equilibration, they were frozen in a 2:1 ratio of 20% sucrose:optimal cutting temperature compound (Tissue Tek, Miles Inc., Elkhart, IN, United States). Frozen brain samples were then sectioned horizontally using a cryostat (Leica Biosystems, Wetzlar, Germany) at a 25 μm thickness throughout the entire depth of the implant (∼1600 μm).

Brain tissue sections between 150–300 μm cortical depth (layer II/III) were chosen for immunohistochemical analysis, corresponding with the depth of observation during 2P imaging. Before staining, frozen tissue sections were re-hydrated with two washes of 1x PBS for 5 min each. For antigen retrieval, slides were incubated in 0.01 M sodium citrate buffer for 30 min at 60 °C. Slides were then incubated in in a peroxidase blocking solution (10% v/v methanol and 3% v/v hydrogen peroxide in 1x PBS) for 20 min on a table shaker (60 r.p.m.) at room temperature (RT) to block for active aldehydes and reduce the chance of non-specific binding. Sections were then pre-treated with a solution of 1% triton X-100 and 10% donkey serum in 1x PBS for 1 h at RT followed by blocking endogenous mouse immunoglobulin G (IgG) with donkey anti-mouse IgG fragment (Fab) for 2 h at 1:10 dilution at RT. Then, sections were rinsed with alternating washes of 1x PBS and 1x PBS-T (1% v/v of Tween-20 in 1x PBS) for four times 4 min each. Primary antibodies were diluted in solution of 1% triton X-100 and 10% donkey serum and applied to slides for 12–18 h at 4 °C. All primary antibodies used in this study are listed in table ​table1.1. Sections were rinsed in 3 × 5 min washes of 1x PBS the following day. Secondary antibodies were diluted 1:500 in 1x PBS and applied to slides for 2 h at RT. Secondary antibodies used were: Alexa Fluor 488 donkey anti-mouse, Alexa Fluor 488 donkey anti-rabbit, Alexa Fluor 568 donkey anti-mouse, Alexa Fluor 568 donkey anti-rat, Alexa Fluor 568 donkey anti-goat, Alexa Fluor 568 donkey anti-sheep, Alexa Fluor 647 donkey anti-mouse, Alexa Fluor 647 donkey anti-chicken, and Alexa Fluor 647 streptavidin (Abcam, Cambridge, UK). Then, slides were rinsed once more with 3 × 5 min washes of 1x PBS. To stain for cell nuclei, slides were incubated in Hoechst 33 342 (Invitrogen) at 1:1000 in 1x PBS for 10 min at RT followed by another rinse 3 × 5 min with 1x PBS. Finally, slides were cover slipped using Fluoromount-G (Southern Biotech, Birmingham, AL, United States) and sealed prior to imaging.

List of primary antibodies used for immunohistochemical staining.

Samples were imaged using a confocal microscope (FluoView 1000, Olympus, Inc. Tokyo, Japan) using a 20x oil-immersive objective lens (Nikon Instruments, Melville, NY). For each section, an ipsilateral and contralateral image was captured using same laser intensity and image settings for data normalization. For each image, a z-stack was collected consisting of six image planes each spaced 5 μm apart (635.9 × 635.9 μm, 1024 × 1024 pixels on FV10-ASW Viewer V4.2). Raw images were saved as 16-bit grayscale TIFFs.