View source for DMU Acetyl-Histone and c-Fos Immuno

Test of role of sex in LiCl-induced and butyrate-enhanced acetylation of histones and c-Fos expression as measured by flurorescent immunohistochemistry.

Dose of NaB and LiCl and timing based on Figure 7 of [https://houptlab.org/papers/pdfs/Houpt065.pdf Kwon and Houpt, Brain Research, 1333 (2010) 36–47], PMID: 20346924 [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2871962/ PMCID: PMC2871962] 

First Injection:
* 0.15 M LiCl (6 ml/kg)
* 0.15 M NaCl (6 ml/kg)

Second Injection:
* 0.3 M Na Butyrate = 3.3g/100ml water (12 ml/kg)
* 0.3 M NaCl = 18g/liter water (osmotic control) (12 ml/kg)

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==Data== 


==People== 

Durba Mukherjee



==Agenda==

'''Rats arrive''' (2022-02-23)

male (n=8) DOB: 2021-12-27

female (n=8) DOB: 2021-12-6 


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'''Groups'''

4 groups for each sex: NaCl-NaCl, LiCl-NaCl, NaCl-NaB, LiCl-NaB 

Female: DMU01-DMU08; Male: DMU09-DMU16

* NaCl-NaCl: DMU 1,5,9,13
* LiCl-NaCl: Female: DMU 2,3,7; Male: DMU 10,11,15
* NaCl-NaB:
* LiCl-NaB: Female: DMU 4,6,8; Male: DMU 12,14,16




'''Injections and Perfusion '''

Rats weighed and given first injection (NaCl or LiCl). 10 minutes later, second injection (NaCl or NaB). 50 minutes later, overdosed with pentobarbital and transcardially perfused, first with 100 ml 0.15 M NaCl/NaNitrite/heparin, then 400ml of 4% PFA. Brain dissected, forebrain/brainstem blocked, post-fixed in 4% PFA for 1 h, then cryopotected in 30% sucrose for 2-3 days.



2022-2-26: DMU01-04,DMU9-12 perfused
2022-2-27: DMU05-8,DMU13-16 perfused

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'''Brain Sectioning'''

36 x 40 micron sections through hindbrain (NTS)

2022-3-2: DMU01-04,DMU9-12 hindbrain sectioned
2022-3-3: DMU05-8,DMU13-16 hindbrain sectioned

'''Immunohistochemistry'''

Usual IHC protocol:  30 min Triton, overnight primary, 1 h secondary, mount with VectaShield w/DAPI. 

anti-acetyl-Histone Primary: anti-acetyl-Histone H3 (Lys14) rabbit polyclonal, Millipore 07-353, Lot #3259085, r. 2019-09-26 (use at 1:2000)
Secondary: FITC conjugated Goat Anti-Rabbit, Jackson Labs 111-095-144, Lot #77909, r. 2008-05-24 (use at 1:100)

anti-c-Fos Primary: anti-c-Fos monoclonal antibody (E-8), Santa Cruz sc-166940, Lot # L1621, r. 2022-3-3 (use at 1:100)
Secondary: Texas Red conjugated Goat Anti-Mouse, Jackson Labs 115-075-146, Lot #72021, r. 2008-05-24 (use at 1:100)

2022-3-3 thru 2022-3-4: DMU01-DMU16 hindbrain sections processed (12 sections each, 500 λ/well).
 
 Dilutions
 16 wells x 500 λ = 8000 λ, so make 9000 λ
 
 Primaries:
 9000 λ PBS/BSA
 anti-acetyl-Histone at 1:2000 => 9000/2000 = 4.5 λ
 anti-c-Fos at 1:100 => 9000/100 = 90 λ
 
 Secondaries:
 9000 λ PBS/BSA
 anti-Rabbit at 1:100 => 9000/100 = 90 λ
 anti-Mouse at 1:100 => 9000/100 = 90 λ





[[Category:Taste Aversion]]
[[Category: CTA Experiments]]