TITLE: Quantification of pathological protein accumulation (aSyn and tau) in transplanted human iPSC-derived dopamine neurons
AUTHORS: Giselle Sagredo, Hongyun Li, YuHong Fu, Glenda Halliday

[DESCRIPTION]
This protocol details the immunohistochemistry protocol and subsequent imaging analysis protocols for quantifying pathological protein accumulation in human derived iPSC dopaminergic neurons transplanted into athymic mice. Tissue sections were stained free floating for human dopamine cells (tyrosine hydroxylase antibody) and pathological markers (Table 1) as per protocol.

[BEFORE_START]
For qualitative and quantitative analyses of iPSC-derived dopamine neurons, images of whole brain sections and higher magnification images of dopamine cells were captured using an Olympus VS200 slide scanner and a Nikon C2 confocal microscope. 
 
 Olympus VS200 slide scanner: For both qualitative and quantitative analyses of iPSC-derived dopamine neurons, images were captured at 20x magnification with a z-range and z-spacing of 12µm and 1.5µm, respectively, and at 60x at one z-plane on the slide scanner.

Nikon C2 confocal microscope: For z-stack qualitative analyses of iPSC-derived dopamine neurons,
images were captured at multiple magnifications on the confocal microscope. A 10x overview image of the regions of interest (ROI) was captured, followed by multiple magnifications (60x-120x) of the ROI to capture cell morphology and any potential pathologies. Z-stacks at 120x (step size= 0.3µm) were also
performed followed by Huygens deconvolution using Huygens Professional version 23.04 (Scientific Volume Imaging, The Netherlands, http://svi.nl) for image presentation. Parameters including laser, gain and offset were selected based on single channel labelling for each section. 

For image analysis, FIJI, v. 2.9.0/1.53t (1), and QuPath, V 0.5.0 (2), software were used.



[STEPS]
SECTION: Tissue processing and immunofluorescence staining
1. Day 1
Tissue Prep
Pour sections into well insert in well-plate to separate the cryoprotectant storage solution from
the sections. 
Move well-insert to well-plate containing approximately 6 mLof 1 x phosphate buffered saline (PBS)
in each well. 
Wash sections for 30 min in 1 x PBS on an orbital shaker changing the buffer every 5 min


SECTION: Tissue processing and immunofluorescence staining
2. Antigen retrieval

       Place sections in glass vials containing 6 mLof 10 millimolar (mM) sodium citrate buffer (pH 6.0). 
       Place in the steamer at 98 °Cfor ~20 min. 
       Cool sections to room temperature and transfer back to corresponding wells. 
       Wash sections in 1 x PBS for 5 min and repeat. 








SECTION: Tissue processing and immunofluorescence staining
3. Blocking and primary antibody incubation
Incubate sections in blocking buffer (containing 2% donkey serum, 1% bovine serum albumin solution, 0.2% Triton X-100, 0.1% gelatin and 0.1% Tween-20 in PBS (PBST)) for 2 hours on an orbital shaker at room temperature.
Incubate sections in the cocktail of primary antibodies in blocking buffer as per Table 1 for ~72 hours at 4°C on the orbital shaker.
SECTION: Identification of protein pathologies
6. Dopamine neurons were identified in scanned images and manually counted based on TH immunoreactivity together with a large dull nucleus (Fig. 1 and 2). Aggregate-like structures were identified, counted and analysed in 3D image reconstructions if phosphorylated a-synuclein (pS129)
immunofluorescence appeared within or partially within TH+ neuronal somata.
Similarly, aggregate-like structures labelled by phosphorylated tau (AT8) immunofluorescence were identified and further analysed in 3D images if they appeared within or partially within TH+ neuronal somata (Fig. 2). PS129 and AT8 tau immunofluorescence labelled morphologically diverse structures in neurons (Fig. 1 and 2). 


 

 

SECTION: Tissue processing and immunofluorescence staining
4.  
Day 2 
Secondary antibodies
Wash sections in 0.1% PBST for30 min, changing the buffer every 10 minutes. 
Incubate sections in corresponding secondary antibodies (Table 1) and bisbenzimide H 33342 trihydrochloride (Hoechst, Cat.# B2261; Sigma-Aldrich) in blocking buffer for120 min at Room temperature on the orbital shaker.
Wash sections in 0.1% PBST for 30 min, changing the buffer every 10 min. 
Wash sections in 1 xPBS for 15 min, changing the buffer every5 min.





SECTION: Tissue processing and immunofluorescence staining
5. Mounting and sealing
Place sections in a petri dish containing 1 x PBS and immerse slide at an angle to gently mount sections
onto slides using a brush. 
Place the coverslip gently on the slide using anti-fade fluorescence mounting medium (Cat.# S3023, DAKO). 
Once dry, seal slides with CoverGrip coverslip sealant (Cat.# 23005, Biotium) and store slides away from light at 4 °C.

SECTION: Imaging and analyses - QuPath analysis
7. Contour
Using the polygon annotation tool, contour the ROI based on pan a-syn and pS129 staining together with the outline observed from the camera images captured prior to staining. The ROI contour provides the area (µm2) of TH+ signals, which were used to detect total cell number (Hoechst), TH+ cell number, and intensity data as outlined below.

SECTION: Imaging and analyses - QuPath analysis
9. Intensity data and masking

 Extract intensity data from the ROI contour using the “add intensity features” function.
 Create thresholders using different intensity parameters guided by the detection measurements provided for the Cell: TH mean and standard deviation to generate masks within the ROI annotation. 

a.       Generate the first mask of the TH+ area fraction including all TH immunoreactivity within the ROI. 
b.       Generate the second mask utilized a much higher intensity to mask mainly the TH+ cells and major processes or projections. 
c.       Add intensity features to the masks created for the detection of mean intensities of other channels within the TH+ masks.
SECTION: Imaging and analyses - QuPath analysis
8.  
Cell detection and other counts

Annotate a small region close to the ROI contour to test the accuracy of the cell detection parameters using the Hoechst channel, in different areas to account for different nuclei intensities and morphologies. Once the parameters (nucleus parameters including sigma, minimum and maximum area, intensity, and cell expansion) are used to result in a count within 10% variance of a manual count, these parameters may then be applied to the ROI annotation for cell detection. 
Using the acquired parameters, apply the cell detection tool to the ROI to identify the total cell number. 
Using the point tool, manually count the TH+ neurons and create an annotation. 
Using the point tool, manually count the TH+ neurons with pS129 immunofluorescence (see definition
above) by toggling off individual channels together with the counted “TH+ cells point annotation”. 
SECTION: Statistical analyses
10. After identifying all TH+ neurons in the ROI, the TH+ cell proportion, density, projections and cell size were calculated. Non-parametric statistics were performed using Kruskal-Wallis tests and post-hoc Dunn’s multiple comparisons tests (Prism 10 (v.10.0.0)) to identify any differences in the above parameters between groups with p < 0.05 as the level of significance.