Figure 1. Demonstration of the automated counting process. A first macro creates a Region of interest (ROI) by automatically setting a threshold for TH positive areas (A). A second macro then lays the ROI over the corresponding peripherin stained image and creates masks wherever TH and peripherin staining are co-located (B, C). These masks are then counted and a list is displayed, showing the counts for each image that was analysed. A merge image is shown to illustrate the co-localization (D). White arrows indicate fibers that can be identified by eye, green arrows indicate those identified by co-localization only.

Materials and Reagents

1. Pipette tips (Starlab, TipOne 1 ml, 200 µl, 10 µl, catalog numbers: S1111-6001, S1111-0000, S1111-3000)
2. Microscopy cover glass (Engelbrecht, Cover Glass 24 mm x 60 mm, catalog number: K12460)
3. Microtome blades (Feather, Disposable Microtome Blade, catalog number: C-35)
4. Specimen molds (Sakura Finetek, Tissue-Tek Cryomold Intermediate 15 mm x 15 mm x 5 mm, catalog number: 4566)
5. Microscopy slides (VWR, Adhesion slides SuperFrost Plus 75 mm x 25 mm, catalog number: 631-0108)
6. PAP pen (Thermo Fisher, Super PAP Pen, catalog number: 008899)
7. Erlenmayer flask (Brand, Erlenmayer flask 250 ml, catalog number: 92736)
8. C57B/6J mice (Janvier Labs, C57B/6JRj, catalog number: C57B/6JRj)
9. Goat Serum (NGS) (Agilent Dako, Goat Serum (Normal), Whole serum, 10 ml, catalog number: X090710-8), store at 4 °C
10. Rabbit anti TH antibody (Merck, Anti-Tyrosine Hydroxylase Antibody, catalog number: AB152), store at -20 °C
11. Chicken anti Peripherin antibody (Abcam, Anti-Peripherin antibody, catalog number: ab39374), store at -20 °C
12. Goat anti Rabbit antibody (Invitrogen, Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594, catalog number: A-11037), store at 4 °C
13. Goat anti Chicken antibody (Abcam, Goat Anti-Chicken IgY H&L (Alexa Fluor^® 488), catalog number: ab150169), store at 4 °C
14. Embedding Medium (Sakura Finetek, Tissue-Tek^® O.C.T.^TM Compound, 12 x 125 ml, catalog number: 4583), store at room temperature (RT)
15. Triton X (Sigma-Aldrich, Triton ^TM X-100, catalog number: T8787), store at RT
16. Paraformaldehyde (PFA) (Sigma-Aldrich, Paraformaldehyde, reagent grade, crystalline, catalog number: P6148), store at 4 °C
17. Hydrochloric acid (Sigma-Aldrich, Hydrochloric acid 36.5-38.0%, BioReagent, for molecular biology, catalog number: H1758), store at RT
18. Sodium Hydroxide (Merck, Sodium hydroxide 1.000 l, catalog number: 1099130001), store at RT
19. Prolong Gold (Invitrogen, ProLong^TM Gold Antifade Mountant with DAPI, catalog number: P36931), store at 4 °C
20. Phosphate buffered Saline (Gibco, PBS (10x), pH 7.4, catalog number: 70011044), store at RT
21. Liquid nitrogen (Linde, CRYOSPEED MED, catalog number: 4130919)
22. Deionized H₂O
23. 3% PFA (250 ml) (see Recipes)
24. PBS 1x (see Recipes)
25. Blocking solution (see Recipes)
26. Washing solution (see Recipes)
27. Secondary antibody diluent solution (see Recipes)
    

Equipment

1. Cryostat (Leica, CM3000 Research Cryostat)
2. Microscope (Zeiss, Axioskop 2 plus)
3. Camera (Nikon, DSV Vi1)
4. PC (Intel Core i3-7100U CPU, 2.40 GHz, 8.00 GB RAM, Windows 10 Enterprise, 64-bit, Intel HD Graphics 620)
5. Magnetic stir heater (IKA, IKAMAG, catalog number: RET)
6. -80 °C freezer (Thermo Scientific, Forma 906, catalog number: 906)
7. Electronic scale (Sartorius, electronic precision scale, catalog number: 1465 MP8)
8. Shaker (Peqlab, Mini Rocker-Shaker, catalog number: MR-1)
9. Micro pipettes (Eppendorf, Research plus variable 0,1-1 ml, 20-200 µl, 2-20 µl, 0,5-10 µl, catalog numbers: 3123000063, 3123000055, 3123000039, 3123000020)
   

Software

1. ImageJ (NIH, https://imagej.nih.gov/ij/)
2. NIS-Elements (Nikon, https://www.nikon.com/)

Procedure