February 13, 2009

GFAP stain

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T09-4VJBCF8-2&_user=2446496&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000053459&_version=1&_urlVersion=0&_userid=2446496&md5=50f162973cf9f74533b4e6693e83dae8

Primate-specific alterations in neural stem/progenitor cells in the aged hippocampus

 

http://www.nature.com/neuro/journal/v7/n11/full/nn1340.html
GFAP-expressing progenitors are the principal source of constitutive neurogenesis in adult mouse forebrain

Immunohistochemistry of tissue sections.
Frozen tissue sections were prepared of 4% paraformaldehyde-fixed brains17. Bright-field immunohistochemistry was performed using biotinylated secondary antibodies (Vector), biotin-avidin-peroxidase complex (Vector), and diaminobenzidine (brown; Sigma) or Vector blue (Vector) as the developing agents. Fluorescence immunohistochemistry was performed using AlexaFluor tagged secondary antibodies Alexa 488 (green), Alexa 568 (red), Alexa 350 (blue) or Alexa 633 (pseudocolor blue) (Molecular Probes). Primary antibodies used were rabbit anti−HSV-TK (1:10,000; ref. 17); mouse anti-GFAP (1:10,000; Chemicon, MAB3402); sheep anti-BrdU (1:5000; Maine Biotechnology Services, PAB105P); mouse anti−PSA-NCAM (1:2000, Chemicon, MAB5324); goat anti-doublecortin (1:500; Santa Cruz, sc8066); mouse anti-NeuN (1:1000; Chemicon, MAB377); rabbit anti−beta-galactosidase (1:1000; ICN Pharmaceuticals, 55976); rabbit anti−green fluorescent protein (1:1000; Molecular Probes, A-21311); Tuj1 (1: 1000; Covance, MMS-435P); mouse anti-O4 (1:1000; Chemicon, MAB345). 4',6-diamidino-2-phenylindole (DAPI; Molecular Probes, D-1306) was used as a fluorescent counterstain. Sections stained for BrdU were pretreated with 2 M HCl for 30 min and neutralized with PBS before incubation in primary antibody. Stained sections were examined and photographed using bright-field and fluorescence microscopy (Zeiss), and scanning confocal laser microscopy (Leica).


http://www.jneurosci.org/cgi/content/full/23/7/2824
The Predominant Neural Stem Cell Isolated from Postnatal and Adult Forebrain But Not Early Embryonic Forebrain Expresses GFAP

<i>Immunocytochemistry.</i> Cells plated on glass coverslips precoated with PLL (Sigma) were fixed in 4% paraformaldehyde for 30 min and stained by immunofluorescence with the following primary antibodies: anti-Tuj1 (1:1000; Berkeley Antibodies, Richmond, CA), anti-GFAP (1:2000; Dako, Carpinteria, CA), anti-O4 (1:100; Chemicon, Temecula, CA), anti-nestin (Rat401; 1:100; Developmental Studies Hybridoma Bank, The University of Iowa, Iowa City, IA), and anti-herpes simplex virus (HSV)-TK (1:1000) (Bush et al., 1998). Primary antibodies were visualized with Alexa 568- (red), Alexa 488- (green), and Alexa 350 (blue)-conjugated secondary antibodies (Molecular Probes, Eugene, OR). Hoechst 33342 (blue) was used as a fluorescent nuclear counterstain. Stained cultures were examined and photographed by fluorescence microscopy (Zeiss, Oberkochen, Germany). For quantitative analysis, double- or single-labeled cells were counted (a minimum of three coverslips per condition) with image-analysis software (MicroBrightField Inc.). Tissue sections of 4% paraformaldehyde-fixed brains from adult mice were double stained (Bush et al., 1999) with primary antibodies: anti-HSV-TK (1:1000; P. Collins) visualized with an Alexa 568 (red)-conjugated secondary antibody (Molecular Probes), combined with anti-GFAP (Dako) that was conjugated directly with Alexa 488 (green; Molecular Probes). Stained sections were examined and photographed by scanning confocal laser microscopy (Leica, Nussloch, Germany). 

http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=18638414

Successful elimination of non-neural cells and unachievable elimination of glial cells by means of commonly used cell culture manipulations during differentiation of GFAP and SOX2 positive neural progenitors (NHA) to neuronal cells



Protocols of differentiation

 

1. Differentiation of GFAP+ neural progenitors to non-neural cells

GFAP-positive neural progenitors (NHA) were plated on six well dishes or uncoated chambers (Nunc) at a density of 10 000–15 000 cells/cm2 and passaged in expansion medium containing 3% FBS, rhEGF, insulin and ascorbic acid (Cambrex AGM Bullet Kit). After every passage, the cells were cultured for 5 days before the next passage was performed.

 

 

2. Differentiation of GFAP+ neural progenitors to neuronal and glial populations

GFAP-positive neural progenitors (NHA) at the passage 0 were plated at a density of 10 000–15 000 cells/cm2 on Matrigel thin-coated (according manufacturer's protocol, BD Biosciences), chamber slides and cultured in expansion medium (Cambrex AGM Bullet Kit) until reaching confluence. After reaching confluence, the medium was changed to N2- or B27-supplemented DMEM F12 (Invitrogen). A final concentration of 10 ng/ml of growth factor was achieved with the optional addition of either bFGF – basic fibroblast growth factor, SHH – sonic hedgehog or GDNF – glial derived neutrophic factor (Invitrogen). The cells were then cultured under differentiation conditions for 5–7 days, with medium replacement every 2 days.

 

 

3. Differentiation of GFAP+ progenitors to neuronal, glial and non-neural cells

GFAP+ neural progenitors were plated on six well dishes or uncoated chambers at a density of 10 000–15 000 cells/cm2 and kept for one to two passages in expansion medium (Cambrex AGM Bullet Kit). After every passage, the cells were kept for 5 days before the next passage was performed. After reaching confluence, the medium was changed to N2- or B27-supplemented DMEM F12 (Invitrogen). A final concentration of 10 ng/ml of growth factor was achieved with the optional addition of one of the following growth factors: bFGF, SHH or GDNF (Invitrogen). The cells were then cultured under differentiation conditions for 5–7 days, with medium replacement every 2 days.

0推薦此文章
Today's Visitors: 0 Total Visitors: 37
Personal Category: Uncategorized Articles Topic: feeling / personal / murmur
歷史上的今天:
[Trackback URL]

Reply
  • 1樓

    1樓搶頭香

    有暴力傾向的 都可以交給神經外科做對照組 查一下:1.peptide進出nuclear pore的染色圖
    片,2.腦壓血壓體溫基本生命徵象,3.neurotransmitters的密度,4.當然與記憶發明相關的
    peptides也都可以觸類旁通.

  • H.H.Li at July 8, 2012 10:53 PM comment | email Homepage
Post A Comment









Yes No



Please input the magic number:

( Prevent the annoy garbage messages )
( What if you cannot see the numbers? )
Please input the magic number

誰來收藏
Loading ...
unlog_NVPO 0