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Turner et al. LPS preconditioning neuroprotective

tube, striking the metal disk. The disk was then antibody (AbD Serotec, Kidlington, UK) at a dilution
removed while the rat was under anesthesia, the of 1:100 in 4% horse serum in DPBS overnight.
skull inspected, and the wound sutured. The animal Sections were washed three times in DPBS
was then returned to its cage, which was placed on and incubated in a biotinylated anti-mouse IgG
a heating pad. Recovery from injury and anesthesia secondary antibody (Vector Laboratories) diluted
were monitored. No mortality was observed with at 1:10,000 in 4% horse serum in DPBS for 4 h.
the current injury parameters, and no gross lesions Following secondary antibody incubation, tissues
were apparent at the time of sacrifice indicating mild were incubated in alkaline phosphatase (Life
diffuse axonal injury. Technology, Carlsbad, CA) diluted at 1:100 in Tris-
bovine serum albumin for 1 h. Then, tissues were
Tissue preparation rinsed three times in DPBS and incubated in Fast
Seven days after TBI, animals were anesthetized Blue BB salt (Santa Cruz Biotechnology, Santa
and perfused transcardially with physiological saline. Cruz, CA) for 5 min. Tissues were washed in xylene,
Brains were removed and sectioned for histological mounted using an antifade agent, and cover slipped.
analysis. The frontal cortex was selected for The slides were sealed with acrylic and stored in the
histological analysis. Tissue sections were placed dark in a laboratory refrigerator.
in 4% paraformaldehyde for a minimum of 1 week.
Following fixation, brains were processed using GFAP and OSMR staining
a Tissue-Tek VIP 5 Automatic Tissue Processor Tissues were labelled with rabbit against anti-cow
(Sakura Finatek, Torrence, CA). Processed tissues GFAP (DAKO) antibody at a dilution of 1:500 in
were paraffin-embedded with Tissue-Tek Tec 5 5% horse serum in PBS overnight at 4 °C. Next,
embedding system (Sakura Finatek, Torrence, CA) sections were washed twice for 10 min each in
and sliced (6 µm) using a Leica RM2235 microtome PBS prior to application of Alexa Fluor 488 goat
(Leica Microscopes, Buffalo Grove, IL). Sections anti-rabbit IgG (Invitrogen, Grand Island, NY) at a
were mounted on glass slides and heat-fixed. dilution of 1:100 in PBS for 3 h. Following secondary
Immediately prior to staining, tissues were de- antibody incubation, slides were rinsed twice for
paraffinized with xylene and alcohol washes. [14] 10 min each in PBS. Then, tissue was labelled
Fluoro-Jade B (FJB) staining with a goat anti-mouse OSMR antibody (LifeSpan
Biosciences, Seattle, WA) at a dilution of 1:200 in
FJB staining was used to identify neural degeneration.
For FJB labelling, slides were rehydrated through a 5% horse serum in PBS overnight at 4 °C. Following
series of alcohol and deionized (dH O) water rinses incubation, slides were rinsed twice for 10 min each
2
then incubated in 0.06% potassium permanganate in PBS prior before applying biotinylated anti-goat
for 10 min. Then, slides were rinsed for 2 min in IgG (Vector Laboratories) at a dilution of 1:10,000
dH O water and incubated with FJB in 0.1% acetic in 5% horse serum in PBS for 2 h. Next, slides were
2
acid for 20 min. After staining, slides were washed rinsed twice for 10 min each in PBS and incubated
three times in dH O. in Streptavidin Alexa Fluor 546 (Life Technology)
2 at a dilution of 1:100 in PBS for 1 h. Slides were
GFAP staining rinsed in PBS for 10 min and then coverslipped
Tissues were incubated in rabbit anti-cow GFAP with Vectashield Mounting Media containing DAPI
antibody (Dako, Glostrup, Denmark) at a dilution of (Vector Laboratories). Finally, slides were sealed
1:500 in 4% horse serum in Dulbecco’s phosphate with acrylic and stored in the dark in a laboratory
o
buffered (DPBS) overnight. Then, tissues were refrigerator at 4 C. Images were acquired using
washed three times in DPBS and incubated in a Zeiss Axio Imager 2 microscope and quantified
biotinylated anti-rabbit IgG antibody (Vector using ImageJ with standard co-localization
Laboratories, Burlingame, CA) diluted at 1:10,000 quantification techniques and the co-localization
in 4% horse serum in DPBS for 4 h. Next, tissue was plugin established by Bolte et al. [15]
treated with avidin D-horeradish peroxidase (Vector
Laboratories, Burlingame, CA) diluted at 1:1,000 in Histological quantification
DPBS for 1 h. Sections were then stained in DAB Stereology and optical fractionation were used
chromogen solution (Vector Laboratories) for 5 min, to quantify histological results as previously
then tissues were rinsed three times in DPBS and described. [16-18] Briefly, a region of interest
dried overnight. encompassing the corpus callosum was drawn at
low power using an Olympus AX70 microscope
M1 microglia staining and StereoInvestigator software. The region
Tissues were incubated in mouse anti-rat CD68 encompassing the corpus callosum was chosen
8 Neuroimmunology and Neuroinflammation ¦ Volume 4 ¦ January 20, 2017