Author Topic: Problem with morphology of Spinal cord free floating section after AR  (Read 6260 times)

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Offline sreenivas1234

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Hi,

I performed AR using 0.05% Citraconic anhydrate 95C for 45-minutes on Mouse Spinal cord 60 um thick sections. after 45-minutes all the tissue samples are dehydrated and became like ball like rounded structure. totally the spinal cord structure was lost. and after staining there is huge background noise.

the protocol i followed:
0.05% CA for 45 minutes at 95 C
wash with 0.2% triton-x 100 in 1M PB (15 minutes 3 washes)
added primary antibody in 0.5% tritonx 1MPB incubated for O/N
wash with 0.3% triton X 100 1M PB 3 times 15 minutes each
Secondary antibody in 0.2% Triton in 1M PB O/N
wash with 0.3% Triton x100 1MPB 3 times

DAPI staining
Mount them with Flourogold

please help me



Offline sreenivas1234

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Re: Problem with morphology of Spinal cord free floating section after AR
« Reply #1 on: November 03, 2010, 06:13:57 AM »
i forgot to mention the PH of Citraconicn anhydrate solution is 7.4 exactly.
Adjusted with 5M HCL and 5M NaOH solution

Offline excalibur

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Re: Problem with morphology of Spinal cord free floating section after AR
« Reply #2 on: November 03, 2010, 01:36:16 PM »
If you are working with frozen sections, AR is an unneccessary step.

If working with paraffin, you can lower the AR temp and keep it in for a longer time. Cover the container to avoid evaporation.
Paula K. Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
8901 S Santa Fe, Suite G
Oklahoma City, OK 73139
405-759-3959
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Offline sreenivas1234

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Re: Problem with morphology of Spinal cord free floating section after AR
« Reply #3 on: November 03, 2010, 03:57:06 PM »
If you are working with frozen sections, AR is an unneccessary step.

If working with paraffin, you can lower the AR temp and keep it in for a longer time. Cover the container to avoid evaporation.

thanx for your reply.

I not using frozen sections. the mice are perfused with 4% Formaldehyde. spinal cord changed into 15% 24hr 30% sucrose 24hr made 60um sections using microtome.
kept in PBS with azide.

I cover the water bath with aluminum foil and before placing the tissue in citraconic anhydrate solution i checked the temp with normal mercury thermometer (95C) then I will insert the sections.

I will try to reduce the temperature and perform the same protocol.

I used 0.1M PB I had written it wrongly 1M in above post. please ignore that.

Re: Problem with morphology of Spinal cord free floating section after AR
« Reply #3 on: November 03, 2010, 03:57:06 PM »