Inhibitory Interneuron Cocktail
Index
2 color coktail re-imaged - June 28 2012
2 color cocktail - June 18 2012
24 hour cocktail - June 18 2012
48 hour cocktail, lo/hi - June 18 2012
competition between 2°Ab - June 15 2012
donkey serum conc - June 14 2012
5-Ab cocktail first attempt - June 11 2012
Trial III.3 June 28 2012
"2 color cocktail re-imaged"
Purpose: to image distinct channels without excitation of the wrong fluorescence
Slices from Trial III.2 were re-imaged on the confocal scope; imaging each channel one at a time (rather than having both PMTs active at the same time).
image: [.jpg] [.tif] - overlay
[image] - red channel only
[image] - green channel only
Trial III.2 June 18 2012
"2 color cocktail"
Purpose: to visualize differences in staining signal intensity between antibodies raised in different species
2 50-micron sections from animal v23 were washed 3x5 min in PBS and then incubated in 0.3% Triton X-100 (3% from new 10% stock solution) and 1% normal donkey serum for 2 hours on a shaker at room temperature. Sections were then washed 1x10 min in PBS and then incubated in the following primary antibody solution (diluted in PBS) overnight (from 6:00pm on 6/18/2012 to 11:45am on 6/19 [~18 hrs]) at 4C on a shaker:
Rabbit anti Neuropeptide Y (Abcam ab30914, 1:400),
Rabbit anti Somatostatin (Abcam ab103790, 1:200),
Mouse anti Calbindin (Abcam ab82812, 1:400),
Rabbit anti Calretinin (Millipore ab5054, 1:400),
and Mouse anti Parvalbumin (Millipore MAB1572, 1:400).
Sections were washed 3x5 min in PBS then incubated in 488 - Donkey anti Rabbit (Invitrogen A21206, 1:200) and 546 - Donkey anti Mouse (Invitrogen A11036, 1:200) for 2 hours at room temp on a shaker. Sections were washed 3x5 minutes in PBS then mounted on a slide with Fluoromount-G.
[image] - overlay
[image] - green channel only
[image] - red channel only
Trial III June 18 2012
"24 hour cocktail"
"48 hour cocktail"
Purpose: to deduce why antibody signal in cocktails is weaker / less-dense than individual antibody staining; addressing issues of both (1) incubation time and (2) antibody concentration
*note: a new 10% Triton X-100 / PBS stock was made
*note: Since there is only one shaker (which was initially in use), these sections were not on a shaker for the first 4 hours and last 2 hours of incubation.
(1)
4 50-micron sections from animal v23 were rinsed 3x5 minutes in PBS then incubated in a blocking solution of 1% normal donkey serum and 0.3% Triton X-100 (3% of a new 10% stock) for 2:00 hrs at room temp on a shaker.
Sections were rinsed 1x5 min in PBS then incubated in the following primary antibody solution: Rabbit anti Neuropeptide Y (Abcam ab30914, 1:400),
Rabbit anti Somatostatin (Abcam ab103790, 1:200),
Mouse anti Calbindin (Abcam ab82812, 1:400),
Rabbit anti Calretinin (Millipore ab5054, 1:400),
and Mouse anti Parvalbumin (Millipore MAB1572, 1:400) at 4C in PBS, beginning at 1:00pm on 6/18/2012. 2 sections will be incubated for a 24 hour period, the 2 other sections will be incubated for a 48 hour period.
The "24-hour" sections, after approx. 25 hours of incubation in primary antibody, were then rinsed 3x5 min in PBS and incubated in 488 - Donkey anti Rabbit (Invitrogen A21206, 1:200) and 488 - Donkey anti Mouse (Invitrogen A21202, 1:200) in PBS for 2 hours at room temp on a shaker. After, sections were washed 3x5 min in PBS then mounted on a slide with Fluoromount-G.
[image] - 24 hour cocktail
(2)
4 50-micron sections from animal v23 were rinsed 3x5 minutes in PBS then incubated in a blocking solution of 1% normal donkey serum and 0.3% Triton X-100 (3% of a new 10% stock) for 2:00 hrs at room temp on a shaker. Sections were then rinsed 1x5 minutes in PBS.
2 sections were incubated in a low-concentration primary antibody solution for 48 hours at 4C on a shaker:
Rabbit anti Neuropeptide Y (Abcam ab30914, 1:1200),
Rabbit anti Somatostatin (Abcam ab103790, 1:600),
Mouse anti Calbindin (Abcam ab82812, 1:1200),
Rabbit anti Calretinin (Millipore ab5054, 1:1200),
and Mouse anti Parvalbumin (Millipore MAB1572, 1:1200); in PBS.
[image] - 48 hr low-conc
The 2 other sections were incubated in a high-concentration primary antibody solution for 48 hours at 4C on a shaker:
Rabbit anti Neuropeptide Y (Abcam ab30914, 1:400),
Rabbit anti Somatostatin (Abcam ab103790, 1:200),
Mouse anti Calbindin (Abcam ab82812, 1:400),
Rabbit anti Calretinin (Millipore ab5054, 1:400),
and Mouse anti Parvalbumin (Millipore MAB1572, 1:400); in PBS.
[image] - 48 hr hi-conc
Trial II.2 June 15 2012
Purpose: test for competition between among secondary antibodies raised in different animals
2 50-micron sections from animal v24 were rinsed 3x5 minutes in PBS then incubated in a blocking solution of 1% normal donkey serum and 0.3% Triton X-100 (3% of a 10% stock in PBS) for 2:20 hrs at room temp on a shaker. Sections were then washed 1x5 min in PBS and incubated in primary antibody cocktail overnight (~19 hours) at 4C. One section was incubated in primary antibodies raised in rabbit only:
rabbit anti-calretinin (Millipore ab5054, 1:400)
rabbit anti-somatostatin (Abcam ab103790, 1:200)
and rabbit anti-neuropeptide Y (Abcam ab30914, 1:400); in 0.1M PBS.
The other section, as a control, was incubated in all 5 "inhibitory" antibodies:
rabbit anti-calretinin (Millipore ab5054, 1:400)
rabbit anti-somatostatin (Abcam ab103790, 1:200)
rabbit anti-neuropeptide Y (Abcam ab30914, 1:400)
mouse anti-calbindin (Abcam ab82812, 1:400)
and mouse anti-parvalbumin (Millipore MAB1572, 1:400).
The next day, sections were washed 3x5 min in PBS and incubated in donkey anti-rabbit (1:200) or donkey anti-rabbit and donkey anti-mouse (1:200, both), respectively, for 2 hours at room temp on a shaker, covered with foil. Sections were then washed 3x5 min in PBS and mounted on a slide with Fluoromount-G.
Trial II - June 14 2012
Purpose: to see if a higher concentration of donkey serum reduces background
2 50-microns sections from animal v24 were rinsed 3x5 minutes in PBS. One section was then incubated in a blocking solution of 1% normal donkey serum and 0.3% Tirton X-100 (3% of a 10% stock solution), the other section was incubated in 10% donkey serum and 0.2% Triton X-100; both for 1 hour at room temp on a shaker. After a 1x5 min wash, sections were incubated in 1:400 rabbit anti-calretinin (Millipore ab5054) for 1 hour at room temperature, then washed 3x5 min in PBS. Sections were incubated in donkey anti-rabbit (Invitrogen A21206, 1:200) for 2:25 hours room temp then washed 3x5 min and mounted on slides with Fluoromount-G.
No significant difference noted during imaging on confocal microscope.
Trial I - June 11 2012
Purpose: Test run
2 50-micron sections from animal v24 were rinsed 1x10 min in PBS then incubated in blocking solution of 1% normal donkey serum and 0.3% Triton X-100, in PBS, for 2 hours room temp on a shaker. Sections were then washed 1x5 min in PBS and incubated in a cocktail of primary antibodies: anti-calbindin (Abcam ab82812, 1:400), anti-Neuropeptide Y (Abcam ab30914, 1:400), anti-Somatostatin (Abcam ab103790, 1:200), anti-calretinin (Millipore ab5054, 1:400), and anti-parvalbumin (Millipore MAB1572, 1:400) in PBS overnight (approx. 17 hrs) at 4C. Next day, slices were washed 3x5 minutes in PBS and incubated in 488-conjugated donkey anti-rabbit (Invitrogen A21206, 1:200) and 488-conjugated donkey anti-mouse (Invitrogen A21202, 1:200) in PBS for 2 hours room temperature on a shaker, covered with foil. Sections were then washed 3x5 min in PBS and mounted on a slide with Fluoromount-G.
Confocal imaging showed many cell bodies, but not as densely populated as just calretinin alone. It seems that not all inhibitory interneurons that should be represented are stained.
A z-series was taken over a total depth of approximately 40 microns, of one slice.