TITLE: Immunofluorescence protocol for floating mouse brain sections
AUTHORS: Mary Alice Allnutt

[DESCRIPTION]
This protocol details the immunofluorescence for floating sections.

[STEPS]
SECTION: Sectioning and long-term tissue storage
1. Section the brains at   thickness on Leica cryostat (CM1860).

SECTION: Sectioning and long-term tissue storage
2. Collect the sections in cryoprotectant solution in 12-well plates and store at -20 °C.

SECTION: Sectioning and long-term tissue storage
3. To make 1000 ml cryoprotectant (from IHCworld.com): 
 
          a  Sucrose  300 g    b  Polyvinyl-pyrrolidone (PVP-40)  10 g    c  0.1M PB  500 ml    d  Ethylene glycol  300 ml  

SECTION: Sectioning and long-term tissue storage
4. Wash the sections that are stored in cryoprotectant 3x 10 min prior to beginning and immunostaining protocols.
SECTION: Sectioning and long-term tissue storage
4.1. Wash the sections that are stored in cryoprotectant for 10 min prior to beginning and immunostaining protocols (1/3).

SECTION: Sectioning and long-term tissue storage
4.2. Wash the sections that are stored in cryoprotectant for 10 min prior to beginning and immunostaining protocols (2/3).
SECTION: Sectioning and long-term tissue storage
4.3. Wash the sections that are stored in cryoprotectant for 10 min prior to beginning and immunostaining protocols (3/3).
SECTION: Immunofluorescence protocol for free-floating sections
5. 
 
Equilibration: TBS + 0.1% Tx, 10 min.
SECTION: Immunofluorescence protocol for free-floating sections
6. Penetration: TBS + 0.5% Tx, 20 min.
SECTION: Immunofluorescence protocol for free-floating sections
7. Wash 3x 2 min.
SECTION: Immunofluorescence protocol for free-floating sections
8. Neutralization: 0.3 Molarity (M) Glycine in TBS + 0.1% Tx, 30 min.

SECTION: Immunofluorescence protocol for free-floating sections
9. Wash 3x 2 min.
SECTION: Immunofluorescence protocol for free-floating sections
10. Blocking: 3% goat serum in TBS + 0.1% Tx, 80 min.

SECTION: Immunofluorescence protocol for free-floating sections
11. Wash 3x 2 min.
SECTION: Immunofluorescence protocol for free-floating sections
12. Primary incubation:
SECTION: Immunofluorescence protocol for free-floating sections
13. Wash 3-4x 2 min.
SECTION: Immunofluorescence protocol for free-floating sections
14. Secondary incubation:
SECTION: Immunofluorescence protocol for free-floating sections
15. Wash 3-4x 2 min with TBS + 0.1% Tx and then twice with TBS alone.
16. Quench autofluorescence (optional):
17. Mounting
SECTION: Immunofluorescence protocol for free-floating sections
7.1. Wash for 2 min (1/3).

SECTION: Immunofluorescence protocol for free-floating sections
7.2. Wash for 2 min (2/3).
SECTION: Immunofluorescence protocol for free-floating sections
7.3. Wash for 2 min (3/3).
SECTION: Immunofluorescence protocol for free-floating sections
9.1. Wash for 2 min (1/3).
SECTION: Immunofluorescence protocol for free-floating sections
9.2. Wash for 2 min (2/3).
SECTION: Immunofluorescence protocol for free-floating sections
9.3. Wash for 2 min (3/3).
SECTION: Immunofluorescence protocol for free-floating sections
11.1. Wash for 2 min (1/3).
SECTION: Immunofluorescence protocol for free-floating sections
11.2. Wash for 2 min (2/3).
SECTION: Immunofluorescence protocol for free-floating sections
11.3. Wash for 2 min (3/3).
SECTION: Immunofluorescence protocol for free-floating sections
12.1. Prepare in 1% goat serum in TBS + 0.1% Tx.
SECTION: Immunofluorescence protocol for free-floating sections
12.2. 80 min 4°C, shaking.

SECTION: Immunofluorescence protocol for free-floating sections
13.1. Wash for 2 min (1/4).
SECTION: Immunofluorescence protocol for free-floating sections
13.2. Wash for 2 min (2/4).
SECTION: Immunofluorescence protocol for free-floating sections
13.3. Wash for 2 min (3/4).
SECTION: Immunofluorescence protocol for free-floating sections
13.4. Wash for 2 min (4/4).
SECTION: Immunofluorescence protocol for free-floating sections
14.1. Prepare in 1% goat serum in TBS + 0.1% Tx.
SECTION: Immunofluorescence protocol for free-floating sections
14.2. 1:500 for 240 min at Room temperature.

15.1. Wash for 2 min with TBS + 0.1% Tx and then twice with TBS alone (1/4).

15.4. Wash for 2 min with TBS + 0.1% Tx and then twice with TBS alone (4/4).
15.3. Wash for 2 min with TBS + 0.1% Tx and then twice with TBS alone (3/4).
15.2. Wash for 2 min with TBS + 0.1% Tx and then twice with TBS alone (2/4).
16.1. Dip sections briefly in ultrapure H2O.
16.3. Dip sections in ultrapure water again briefly prior to mounting.
 

16.2. Incubate in CuSO4 buffer (10 millimolar (mM) CuSO4 in 50 millimolar (mM) ammonium acetate buffer, pH 5) for 15 min at Room temperature.

17.1. Fill petri dish with TBS. Have serological pipette and secondary container available.
17.2. Place a clean slide in petri dish diagonally, with label resting against the side of the dish, above the level of the solution, and the rest submerged.
17.3. Using fine detail paint brush, lift floating section and gently adhere to slide near the top of the TBS. Can gently tap the section to get it to stick to the slide, or lift one corner of the section along the slide slightly above the water level to help it stick. 
17.4. Using serological pipette, gently remove a few mL of TBS, leaving the section adhered to the slide. 
17.5. After all sections are mounted, allow excess TBS to dry and then mount using Vectashield mounting medium with DAPI and a glass coverslip.