TITLE: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
AUTHORS: Benjamin David Webster Belfort

[DESCRIPTION]
Pipeline for quantifying olfactory sensory neuron axon terminal expression of AAV-derived TdTomato within olfactory bulb tissue sections. This protocol outlines how to use Imaris to isolate TdTomato (experimental signal) and OMP (control signal) to quantify the extent of OSN transduction following nasal inoculation of AAV.

[STEPS]
SECTION: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
2. After 2 weeks, harvest and process olfactory bulb tissue:
SECTION: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
3. Image OB sections using a confocal microscope, such as a Leica TCS SP8, equipped with a 20x/.75 objective and Leica LAS X software.
SECTION: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
4. To obtain the entire volume of each OB section, collect Z-stacks of ~30-40um per sample at 1024x1024 resolution with 4x line averaging and 2x frame averaging.
SECTION: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
5. Stitch images within the LAS X software.
SECTION: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
7. Analyze OB images using Imaris software (version 10.0):
SECTION: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
7.1. For each image, draw a region of interest over the glomerular layer (excluding the olfactory nerve layer) and generate a volume.
SECTION: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
7.3. Generate volumetric surfaces for each of the individually masked channels, using absolute intensity thresholding to account for per-sample background fluorescence signal.
SECTION: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
7.4. Assess transduction efficiency of each serotype; quantify the total area (um^2) of TdTomato signal and OMP signal within the mask (area is used in place of volume, as the TdTomato signal is a cell-fill while anti-OMP antibody only labels the exterior of the axons). 
SECTION: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
7.5. Calculate the ratio of TdTomato signal area to OMP signal area.
SECTION: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
6. To blind the researchers to the samples being analyzed, image files should renamed and randomly sorted.
SECTION: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
8. Convert the files back into their identifiable names after all Imaris-based quantification is completed.
SECTION: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
2.1. Transcardially perfuse mice with 10mL of ice-cold PBS, follow by 10mL of ice-cold 4% paraformaldehyde.
SECTION: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
2.2. Harvest the olfactory bulbs (OBs), fix in 4% PFA at 4°C overnight, and move to 30% sucrose for 36 hours for cryoprotection. 
SECTION: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
2.4. Block tissue sections in 5% Donkey Serum (1X PBS, 0.1% Triton) for one hour at room temperature. 
SECTION: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
2.3. Freeze OBs in OCT (Fisher HealthCare cat# 23-730-571), section at 40um on a cryostat (Leica CM1860), and collect every third section for analysis.
SECTION: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
2.9. Mount tissue on slides, cover with Fluoromount-G (Southern Biotech cat# 0100-01) and a cover glass, and seal the edges with nail polish prior to microscopy.
SECTION: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
2.8. Incubate tissue with Hoechst (1:1000, Thermo Fisher cat# 62249) for 15 minutes at room temperature, and finally wash one more time with PBST. 
SECTION: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
2.7. Wash tissue three times with PBST.
SECTION: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
2.6. Wash sections three times with PBST (0.1% Triton), followed by an incubatation with secondary antibody (anti-goat, Alexa Fluor 488 Invitrogen cat# A-11055) for 1 hour at room temperature.
SECTION: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
2.5. Wash three times in PBST (0.1% Triton) and incubate with anti-OMP (1:20,000, FUJIFILM Wako Chemicals U.S.A. Corporation cat# 544-10001-WAKO) antibody at 4°C overnight. 
SECTION: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
7.2. Mask each channel individually within the volume, setting pixel intensity outside of the volume to 0.
SECTION: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
1. Perform nasal inoculation of TdTomato-expressing AAV (https://www.protocols.io/view/protocol-for-nasal-lavage-of-aavs-into-mice-cypnxvme).
SECTION: Quantification of AAV Transduced Olfactory Sensory Neuron Terminals in the Olfactory Bulb Glomerular Layer
9.