PMID- 21978484
OWN - NLM
STAT- MEDLINE
DCOM- 20120308
LR  - 20230306
IS  - 1872-678X (Electronic)
IS  - 0165-0270 (Linking)
VI  - 203
IP  - 1
DP  - 2012 Jan 15
TI  - Reduction of autofluorescence at the microelectrode-cortical tissue interface 
      improves antibody detection.
PG  - 96-105
LID - 10.1016/j.jneumeth.2011.09.024 [doi]
AB  - Immunohistochemistry (IHC) remains among the most utilized methods for detection 
      of inflammatory events occurring at the microelectrode-cortical tissue interface. 
      It has further become a standard protocol to quantify the intensity of this 
      resulting fluorescent signal, normalized to "background", as a measurement of the 
      extent of inflammatory events. Unfortunately, several sources of autofluorescence 
      could result in variations in this user-defined "background". Notably, we found 
      that the presence of hemosiderin-laden macrophages (HLMs) at the interface 
      resulted in a variable source of background in both green and red fluorescent 
      channels. The HLM-derived autofluorescence prevented the reproducible detection 
      of presumably low-level antigens at the interface. Here we show that treatment of 
      the native cortical tissue for no less than 10 min, with a minimum of 0.5mM 
      copper sulfate, resulted in at least a 70% reduction in native HLM 
      autofluorescence in both green and red fluorescent channels. In the case of 
      highly expressed antigens, such as glial fibrillar acidic protein (GFAP), 
      treatment of immuno-labeled tissue with copper sulfate reduced tissue background, 
      compared to standard IHC methodology, but did not result in significant 
      differences in the quantification of normalized signal intensity. However, 
      treatment with copper sulfate substantially enhanced the detection efficiency of 
      weakly expressed antigens at the device-tissue interface. This study demonstrates 
      that the inclusion of copper sulfate incubation during IHC tissue preparation 
      significantly reduced HLM-derived autofluorescence, and allowed for more accurate 
      detection and quantification of faintly expressed inflammatory markers at the 
      device-tissue interface.
CI  - Published by Elsevier B.V.
FAU - Potter, Kelsey A
AU  - Potter KA
AD  - Department of Biomedical Engineering, Case Western Reserve University, 2071 
      Martin Luther King Jr. Drive, Wickenden Bldg, Cleveland, OH 44106, USA.
FAU - Simon, Joel S
AU  - Simon JS
FAU - Velagapudi, Bharath
AU  - Velagapudi B
FAU - Capadona, Jeffrey R
AU  - Capadona JR
LA  - eng
GR  - I01 RX000334/RX/RRD VA/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
DEP - 20110929
PL  - Netherlands
TA  - J Neurosci Methods
JT  - Journal of neuroscience methods
JID - 7905558
RN  - 9011-92-1 (Hemosiderin)
RN  - LRX7AJ16DT (Copper Sulfate)
SB  - IM
MH  - Animals
MH  - Cerebral Cortex/immunology/*pathology
MH  - Copper Sulfate
MH  - Electrodes, Implanted/*adverse effects
MH  - Hemosiderin/chemistry
MH  - Image Processing, Computer-Assisted
MH  - Immunohistochemistry/*methods
MH  - Inflammation/*etiology/immunology/pathology
MH  - Macrophages/chemistry/metabolism
MH  - Male
MH  - Microelectrodes/adverse effects
MH  - Microscopy, Fluorescence/*methods
MH  - Rats
MH  - Rats, Sprague-Dawley
MH  - Signal-To-Noise Ratio
EDAT- 2011/10/08 06:00
MHDA- 2012/03/09 06:00
CRDT- 2011/10/08 06:00
PHST- 2011/07/27 00:00 [received]
PHST- 2011/09/13 00:00 [revised]
PHST- 2011/09/21 00:00 [accepted]
PHST- 2011/10/08 06:00 [entrez]
PHST- 2011/10/08 06:00 [pubmed]
PHST- 2012/03/09 06:00 [medline]
AID - S0165-0270(11)00572-3 [pii]
AID - 10.1016/j.jneumeth.2011.09.024 [doi]
PST - ppublish
SO  - J Neurosci Methods. 2012 Jan 15;203(1):96-105. doi: 
      10.1016/j.jneumeth.2011.09.024. Epub 2011 Sep 29.