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 Kwon and Houpt, Brain Research, 1333 (2010) 36–47, PMID: 20346924 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)

People

Durba Mukherjee

Agenda

Rats arrive (2022-02-23)

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

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

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

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