Inhibitory Interneuron + NeuN Immunohistochemistry
Index
troubleshooting - June 18 2012
troubleshooting - June 14 2012
troubleshooting, costaining (1-Ab) - June 12 2012
Neun/Inhib costain test, first attempt - June 11 2012
Trial II.3 June 18 2012
Purpose: troubleshooting problem of lack of signal with NeuN staining
Two tests will be run: (1) One test will include a 1:100 dilution of NeuN with an incubation time of 2 hours at room temperature. (2) Another test will consist of a 1:250 dilution of NeuN overnight at 4C.
(1)
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 (a mistake, did not mean to include, should do no harm) for 2 hours on a shaker at room temperature. Sections were then rinsed 1x5 min in PBS. One section was incubated in mouse anti-NeuN (Millipore MAB377x, 1:100, arrived May 6 / Aug 2 2011); the other in rabbit anti-NeuN (Millipore ABN78A4, 1:100, arrived June 12 2012) both for 2 hours at room temperature on a shaker. After, sections were washed 3x5 minutes in PBS then mounted on a slide and coverslipped with Fluoromount-G.
Imaging with dissection microscope showed very good fluorescence for both sections.
(2)
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 (a mistake, did not mean to include, should do no harm) for 2 hours on a shaker at room temperature. Sections were then rinsed 1x10 minutes in PBS. One section was incubated in mouse anti-NeuN (Millipore MAB377x, 1:250, arrived May 6 / Aug 2 2011); the other in rabbit anti-NeuN (Millipore ABN78A4, 1:250, arrived June 12 2012) both overnight (from 6:00pm on 6/18/2012 to 11:45am on 6/19 [~18 hrs] ) at 4C on a shaker. Sections were rinsed 3x5 min in PBS and mounted on a slide with Fluoromount G.
Imaging with dissection microscope showed very good fluorescence for both sections.
Trial II.2 - June 14 2012
Purpose: test if the ms-NeuN is expired
2 50-micron slices from animal v23 were washed 3x5 minutes in PBS on a shaker and incubated in 0.3% Triton X-100 (3% from 10% stock solution) for 1 hour room temperature, then washed 1x5 min in PBS. One section was then incubated in 1:100 mouse anti-NeuN (Millipore MAB377x, arrived May 6 / Aug 2 2011) and the other in 1:100 rabbit anti-NeuN (Millipore ABN78A4, arrived June 12 2012) for 1 hour room temperature on a shaker. Sections were then rinsed 3x5 min in PBS and mounted on a slide with Fluoromount-G.
During imaging, no expression in either section was seen.
Trial II - June 12 2012
Purpose: test co-staining of ca-binding proteins and NeuN, troubleshoot lack of NeuN signal
2 50-micron cortical slices from animal V24 (p55) were washed 1x12 min in PBS on shaker and incubated in 1% normal donkey serum (aliquot had previously been thawed for prior application) and 0.3% Triton X-100 (from 10% stock) in PBS for 90 minutes. Slices were then washed 1x5 minutes in PBS and incubated in 1:100 rabbit anti-calretinin (Millipore ab5054) for 1 hour, room temp on a shaker, followed by another 3x5 minute wash in PBS. Next, one of the sections was incubated in 1:100 488-conjugate mouse anti-NeuN (Millipore MAB377x) and 1:200 555-conjugated donkey anti-rabbit (Invitrogen A31572) for 2 hours at room temp on a shaker; the other section was incubated only in the NeuN. Sections were then washed 3x5 minutes in PBS and mounted on a slide with Fluoromount-G.
Confocal imaging showed a clear calretinin fluorescence on the one slice, but no NeuN staining on either section. The section that had only NeuN (and no calretinin) staining, it seemed that there were some fibers stained but no cell bodies.
Trial I - June 11 2012
Purpose: Test of co-staining calcium-binding proteins and NeuN
2 50-micron cortical slices from animal V23 (p48) were washed 1x10 min in PBS (on shaker) and incubated in a blocking solution (1% normal donkey serum, 0.3% Triton X-100; in PBS) for 2 hours at room temp on shaker. Note: donkey serum came from an aliquot that had been thawed once before in a previous application. Slices were washed 1x10 min in 0.1M PBS on a shaker, then incubated in 1:100 Anti-Calretinin (Millipore ab5054) and 1:100 NeuN (Millipore MAB377x) in PBS for 1 hour at room temperature on shaker. Slices were then rinsed 3x5 minutes in PBS and incubated in 1:200 donkey-anti-rabbit (546 conjugated) for 2 hours, room temp, on a shaker. Following, slices were washed 3x5 min in PBS and mounted on a slide with Fluoromount-G.
Confocal imaging showed no NeuN signal but clear Calretinin. Will try applying NeuN with the secondary antibody to prevent the possibility of NeuN washing out.