Protocol in short:
Sections were washed (3x10min), treated with target retrieval solution and washed again (5×15 min) Sections were rinsed and incubated in FAB blocking buffer with Donkey anti-mouse for 2 hours.
Sections were then incubated for 5 days with the primary antibody 488-labeled directed towards PV. After 5 days, the sections were incubated with PV, OYSTER 550 LABELED (SYNAPTIC SYSTEMS) and 488-conjugated VVA lectin. Sections were then again washed (6×10 min) and (1×10 min) in DAPI. Finally, the sections were mounted on slides, air-dried and then mounted with cover slips.
For Figure 11 the same protocol is applied, except that here the PV antibody and VVA lectin have been incubated for the same amount of time and thus introduced a longer washing step of VVA lectin, which resulted in less background. Figure 10 and 11 shows that both protocols are suitable for a double staining.
Results:
The staining appear specific and show that the protocol are suitable for a double staining targeting parvalbumin neurons.
On January 5, 2017 Mohammad Baragji, University of Copenhagen wrote:
Double staining targeting Parvalbumine expressing interneurons using VVA lectin.
Primary antibody: PARVALBUMIN, MOUSE MAB CLONE 58E1, OYSTER 550 LABELED [SYNAPTIC SYSTEMS, Cat. 195 011C3].
Lectin: Fluorescein labeled Vicia Villosa Lectin (VVL, VVA) [Vector Labs, Cat: FL-1231].
Protocol in short:
Sections were washed (3x10min), treated with target retrieval solution and washed again (5×15 min) Sections were rinsed and incubated in FAB blocking buffer with Donkey anti-mouse for 2 hours.
Sections were then incubated for 5 days with the primary antibody 488-labeled directed towards PV. After 5 days, the sections were incubated with PV, OYSTER 550 LABELED (SYNAPTIC SYSTEMS) and 488-conjugated VVA lectin. Sections were then again washed (6×10 min) and (1×10 min) in DAPI. Finally, the sections were mounted on slides, air-dried and then mounted with cover slips.
For Figure 11 the same protocol is applied, except that here the PV antibody and VVA lectin have been incubated for the same amount of time and thus introduced a longer washing step of VVA lectin, which resulted in less background. Figure 10 and 11 shows that both protocols are suitable for a double staining.
Results:
The staining appear specific and show that the protocol are suitable for a double staining targeting parvalbumin neurons.