Immunofluorescence Multi-label Protocol for Free-floating Fixed Tissue

Wash sections (6 x 0h 10m 0s) in Dilution Media (DM) (0.2Molarity (M)TBS plus 0.05% volumeTriton X-100).

Wash sections for 0h 10m 0s in DM (1/6).

Wash sections for 0h 10m 0s in DM (2/6).

Wash sections for 0h 10m 0s in DM (3/6).

Wash sections for 0h 10m 0s in DM (4/6).

Wash sections for 0h 10m 0s in DM (5/6).

Wash sections for 0h 10m 0s in DM (6/6).

Endogenous peroxidase inhibition (0h 20m 0s). 0.1Molarity (M) Sodium meta-periodate in TBS.

• 100mL 0.2Molarity (M)Tris-buffered saline (TBS)
• 2.13g Sodium meta-periodate

Wash (2 x 0h 10m 0s) in DM.

Wash for 0h 10m 0s in DM (1/2).

Wash for 0h 10m 0s in DM (2/2).

Serum blocking step (1h 0m 0s incubation):

• 100mL DM
• 3mL Normal Serum (species matching the host of the secondary antibody, e.g. horse, goat)
• 2g Bovine Serum Albumin (BSA)

Incubation in primary antibody (18h 0m 0s- 72h 0m 0s). See antibody catalog for concentration of primary antibody.

• 100mL DM
• 1mL Normal Serum (species matching the host of the secondary antibody, e.g. horse, goat)
• 1g BSA
• 0.5mL Triton X-100
  Note

  Optionally, refrigerate 4°Cto keep antibody stable

Wash (6 x 0h 10m 0s) in DM.

Wash in DM for 0h 10m 0s (1/6).

Wash in DM for 0h 10m 0s (2/6).

Wash in DM for 0h 10m 0s (3/6).

Wash in DM for 0h 10m 0s (4/6).

Wash in DM for 0h 10m 0s (5/6).

Wash in DM for 0h 10m 0s (6/6).

Fluorophore-conjugated secondary antibody incubation. (1h 0m 0s) Concentration of secondary antibody is always 1:200 in solvent.

• 100mL DM
• 1mL Normal Serum (species matching the host of the secondary antibody, e.g. horse, goat)
• 1g BSA

Wash (2 x 0h 10m 0s) in TBS.

Wash for 0h 10m 0s in TBS (1/2).

Wash for 0h 10m 0s in TBS (2/2).

Serum blocking step (1h 0m 0s incubation):

• 100mL DM
• 5% (v/v) Normal Serum (species MATCHING THE HOST OF THE PRIMARY antibody, saturates open binding sites on secondary antibody)
• 1g Bovine Serum Albumin (BSA)
  Note

  DO NOT USE any detergent (i.e. Triton X-100, Tween-20, DM) from this step onward! Detergent will wash away the fragment antibodies!

Oversaturate with Fab antibody against host of primary antibody and from same host species as secondary antibody y. (ex. If primary was mouse and secondary was

goat anti-mouse, you would use a Fa goat a anti-mouse se antibody). Working

concentratio40: 1h 0m 0s ( incubation)

• 100mL TBS
• 40 Fab antibody (base concentration is 1.3 mg/mL for fab-goat anti mouse, so use M1V1 = M2V2 to find V1/X).

Wash (2 x 0h 10m 0s) in TBS.

Wash for 0h 10m 0s in TBS (1/2).

Wash for 0h 10m 0s in TBS (2/2).

Incubation in SECOND primary antibody (18h 0m 0s- 72h 0m 0s). See antibody catalog for concentration of primary antibody.

• 100mL DM
• 1mL Normal Serum (species matching the host of the secondary antibody, e.g. horse, goat)
• 1g BSA
  Note

  Optionally, refrigerate 4°Cto keep antibody stable CAN ALSO ADD THIRD PRIMARY ANTIBODY FROM DIFFERENT SPECIES, IF NEEDED.

Wash (6 x 0h 10m 0s) in TBS.

Wash in TBS for 0h 10m 0s (1/6).

Wash in TBS for 0h 10m 0s (2/6).

Wash in TBS for 0h 10m 0s (3/6).

Wash in TBS for 0h 10m 0s (4/6).

Wash in TBS for 0h 10m 0s (5/6).

Wash in TBS for 0h 10m 0s (6/6).

Fluorophore-conjugated secondary antibody incubation against second (and third, if used) primary antibody. (1h 0m 0s) Use different fluorophores for each secondary. Concentration of secondary antibody is always 1:200 in solvent.

• 100mL DM
• 1mL Normal Serum (species matching the host of the secondary antibody, e.g. horse, goat)
• 1g BSA

Wash (3 x 0h 10m 0s) in TBS.

Wash for 0h 10m 0s in TBS (1/3).

Wash for 0h 10m 0s in TBS (2/3).

Wash for 0h 10m 0s in TBS (3/3).

Store tissue in TBS in the refrigerator at 4°C until mounted.

Control for Fragment antibody (Fab): Control tissue should be processed alongside experimental tissue through Day 2, Step 11. Skip second primary incubation all together (Step 12), and complete Day 3. Check under microscope to ensure there is no co-labeling between the two chosen fluorophores.

Use appropriate +/- controls.