All animal experiments were approved by the local ethics committee (BMWFW-66.009/0155-WF/V/3b/2015). C57Bl/6 wild type littermates were injected at postnatal days P5 and P6 with 50 μg/g 79-6 (Calbiochem – Merck, Darmstadt, Germany) in 100 μl vehicle (peanut oil + 10% ethanol) or vehicle only. Retinas were isolated at P7 and stained with biotinylated isolectin B4 (Vector Laboratories, Burlingame, CA) and streptavidin-AlexaFluor555 conjugate (Invitrogen). Whole-mount retinas were analyzed with a Zeiss LSM780 confocal microscope, Photoshop CS4 and HistoQuest 3.5 software (TissueGnostics, Vienna, Austria).
Streptavidin alexa fluor 555 conjugate
Manufactured by Thermo Fisher Scientific
Sourced in United States
Streptavidin Alexa Fluor 555 conjugate is a fluorescent labeling reagent. It consists of the protein streptavidin conjugated to the Alexa Fluor 555 dye. Streptavidin has a high affinity for the small molecule biotin, allowing the conjugate to be used for the detection and localization of biotinylated biomolecules.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Hpa029874, by Merck Group (2 mentions)
Biotinylated isolectin b4, by Vector Laboratories (1 mentions)
Axiovert 200m, by Zeiss (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
Biotin labeled horse anti mouse antibody, by Vector Laboratories (1 mentions)
Sodium alginate salt, by Merck Group (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Streptavidin hrp dy998, by R&D Systems (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
S32355, by Thermo Fisher Scientific (1 mentions)
Pannoramic midi, by 3DHISTECH (1 mentions)
Streptavidin alexa fluor 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Superfrost slide, by Thermo Fisher Scientific (1 mentions)
Dewax, by Thermo Fisher Scientific (1 mentions)
Fluoromount aqueous mounting medium, by Merck Group (1 mentions)
7 protocols using streptavidin alexa fluor 555 conjugate
1
Retinal Vascularization Assay in Mice
2
Detecting Denatured Collagen in Tissue Sections
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To detect denatured collagen, 7 µm longitudinal cryosections were stained using
col2-3/4m antibody, using a previously described protocol.21 (link)
Briefly, sections were dried for 90 minutes at 37°C, fixed for 5 minutes
in 3.7% 0.1 M phosphate-buffered (pH 7.4) formaldehyde and then rinsed
extensively in a large volume of PBS. Sections were dipped in 0.1% tween PBS
incubated with 1% hyaluronidase (testicular, Type I-s, EC 3.2.1.35,
Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes at 37°C to enhance the
permeability of the extracellular matrix by removing PGs. Afterwards, they were
incubated in 10% normal horse serum for 30 minutes to block nonspecific staining
and incubated overnight at 4°C with 1/20 col2-3/4m antibody. The next day,
sections were incubated in biotin-labeled horse anti-mouse antibody (1/400, IgG
(H + L), produced in horse, Vector Laboratories, Inc., Burlingame, CA, USA) for
1 hour at room temperature. Finally, they were incubated with streptavidin 555
reagent (streptavidin, Alexa Fluor 555 conjugate, Invitrogen, Waltham, MA, USA)
for 30 minutes. To detect nuclei, they were counterstained with 1:1000 DAPI
(Thermo Fisher, Waltham, MA, USA). After each preparation step, sections were
rinsed with PBS. After mounting with mowiol, stained sections were digitized at
10× (Zeiss Axiovert 200M, Carl Zeiss, Oberkochen, Germany).
col2-3/4m antibody, using a previously described protocol.21 (link)
Briefly, sections were dried for 90 minutes at 37°C, fixed for 5 minutes
in 3.7% 0.1 M phosphate-buffered (pH 7.4) formaldehyde and then rinsed
extensively in a large volume of PBS. Sections were dipped in 0.1% tween PBS
incubated with 1% hyaluronidase (testicular, Type I-s, EC 3.2.1.35,
Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes at 37°C to enhance the
permeability of the extracellular matrix by removing PGs. Afterwards, they were
incubated in 10% normal horse serum for 30 minutes to block nonspecific staining
and incubated overnight at 4°C with 1/20 col2-3/4m antibody. The next day,
sections were incubated in biotin-labeled horse anti-mouse antibody (1/400, IgG
(H + L), produced in horse, Vector Laboratories, Inc., Burlingame, CA, USA) for
1 hour at room temperature. Finally, they were incubated with streptavidin 555
reagent (streptavidin, Alexa Fluor 555 conjugate, Invitrogen, Waltham, MA, USA)
for 30 minutes. To detect nuclei, they were counterstained with 1:1000 DAPI
(Thermo Fisher, Waltham, MA, USA). After each preparation step, sections were
rinsed with PBS. After mounting with mowiol, stained sections were digitized at
10× (Zeiss Axiovert 200M, Carl Zeiss, Oberkochen, Germany).
3
Bioconjugation of Polymers for Immunoassay
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All chemicals are used without further purification, unless stated otherwise. Alginate sodium salt, formaldehyde (37%), hydrochloric acid (HCl), dimethyl sulfoxide (DMSO), 3,3’,5,5’-tetramethylbenzidine (TMB), and hydrogen peroxide (30% w/w) were purchased from Sigma Aldrich/Merck, St. Louis, MO, United States. Hyaluronic acid sodium salt (40–65 kDa) was purchased from LifeCore, Kloten, Switzerland. Chitosan (90/30/A1) was purchased from BioLog Heppe, Landsberg, Germany. Poly(ethylene glycol) diamine with molecular weights of 2000, 1000, and 600 g mol−1, as well as poly(ethylene glycol) dialdehyde with a molecular weight of 1000 g mol−1, and carboxylic acid poly(ethylene glycol) biotin with a molecular weight of 1000 g mol−1 were purchased from Creative PEGWorks, Chapel Hill, NC, United States. Tert-butyl isocyanide was purchased from TCI Deutschland, Eschborn, Germany. Streptavidin Alexa Fluor 555 conjugate was purchased from Invitrogen, Carlsbad, CA United States. Streptavidin-HRP DY998 was purchased from R&D Systems, Minneapolis, MN, United States. Deionized water from a Milli-Q water purifier with a resistance of 18.2 MΩ cm was used.
4
Biotinylated cDNA Microarray Hybridization
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Biotinylated cDNA is hybridized overnight (45°C for 18 h) to complementary probe (46–47mer) array in single gasket Agilent cassette (G2534A). The hybridization solution (600 µl) contains 6x SSPE, 0.01 µg µl−1 acetylated BSA, 0.05% Tween 20, 5% formamide and 15 µg biotinylated cDNA targets. Prior to loading, the solution is denatured at 65°C for 5 min and cooled on ice for 2 min. Post hybridization, the gasket is removed from array slide under 1x SSPE, washed twice for 3 min with 1x SSPE and once with 0.25x SSPE solution (under gentle stirring). Next, the array slide is immersed in 30 ml solution containing 4 µg Streptavidin Alexa Fluor 555 conjugate (S32355, Invitrogen), 6x SSPE, 0.1 µg µl−1 acetylated BSA, 0.05% Tween 20 and incubated for 2 h at 16°C. The slide is washed with 1x SSPE followed by 0.25x SSPE (3 min each), dried (spin 10 s) and scanned with Axon GenePix 4000B (multiple 532 PMT settings).
5
Immunohistochemical Analysis of Amyloid-Beta and Iba-1 in Brain Tissue
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Brain paraffin sections were first dehydrated with 70% ethanol for 15 min, then put in citric acid (pH 6.0) for antigen retrieval, thereafter blocked with 3% bovine serum albumin in PBS. Next, sections were incubated with primary antibodies (Aβ42, 1/500, cat#805501, Biolegend; Ibα-1,1/500, cat#016-26461, Wako, Osaka, Japan) in 0.3% Triton X-100 in PBS with 5% blocking serum overnight at 4 °C. Thereafter, the following secondary antibodies (Streptavidin Alexa Fluor 555 conjugate, 1/1000, S32355, Invitrogen, Carlsbad, CA, USA for Aβ42; Alexa Fluor 488 goat anti-rabbit, 1/500, A-11008, Invitrogen, for Ibα-1) were used for incubation for 1 h at RT in the dark, sections were then re-stained with DAPI (cat#D1306, Invitrogen) for 10 min and mounted on slides. Images were scanned using Pannoramic Midi (3D Histech Ltd., Budapest, Hungary).
6
Co-localization of Cathelicidin and M. agalactiae
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To co-localize cathelicidin with M. agalactiae, a double immunofluorescence stain was performed.
Three μm tissue sections were mounted on Superfrost™ slides (Thermo Scientific). Dewaxing, rehydration, and antigen retrieval were conducted in Dewax and HIER (heat-induced epitope retrieval) buffer L pH 6.2 (Thermo Scientific) at 98 °C for 20 min by means of an Electric Vegetable Steamer. Slides were then chilled in milli-Q water for 20 min and washed three times in PBST.
Tissues were blocked with Normal Horse Serum (NHS) (VECTOR) for 30 min and then incubated overnight at 4 °C with a rabbit hyperimmune serum raised against M. agalactiae rP48 (Rosati et al., 2000; (link)Cacciotto et al., 2016) (link). On the following day, the tissue sections were washed with PBS-T, incubated with a broad spectrum biotinylated antibody (Invitrogen) for 15 min, and the signal were revealed by incubating with streptavidin-Alexa Fluor 555 conjugate (Invitrogen), for 45 min at room temperature in the dark. After washing 3 times with PBS, the sections were subjected to a new nonspecific blocking step with NHS and incubated overnight at 4 °C with an anti-CAMP, rabbit polyclonal antibody (Sigma Aldrich HPA029874), diluted 1:1500. The signal was revealed as described above using streptavidin-Alexa Fluor 488 conjugate (Invitrogen).
Three μm tissue sections were mounted on Superfrost™ slides (Thermo Scientific). Dewaxing, rehydration, and antigen retrieval were conducted in Dewax and HIER (heat-induced epitope retrieval) buffer L pH 6.2 (Thermo Scientific) at 98 °C for 20 min by means of an Electric Vegetable Steamer. Slides were then chilled in milli-Q water for 20 min and washed three times in PBST.
Tissues were blocked with Normal Horse Serum (NHS) (VECTOR) for 30 min and then incubated overnight at 4 °C with a rabbit hyperimmune serum raised against M. agalactiae rP48 (Rosati et al., 2000; (link)Cacciotto et al., 2016) (link). On the following day, the tissue sections were washed with PBS-T, incubated with a broad spectrum biotinylated antibody (Invitrogen) for 15 min, and the signal were revealed by incubating with streptavidin-Alexa Fluor 555 conjugate (Invitrogen), for 45 min at room temperature in the dark. After washing 3 times with PBS, the sections were subjected to a new nonspecific blocking step with NHS and incubated overnight at 4 °C with an anti-CAMP, rabbit polyclonal antibody (Sigma Aldrich HPA029874), diluted 1:1500. The signal was revealed as described above using streptavidin-Alexa Fluor 488 conjugate (Invitrogen).
7
Dual Immunofluorescence of Cathelicidin and Cytokeratin
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To co-localize cathelicidin and cytokeratin, tissue sections were subjected to a double immunofluorescence, as described above, with slight modifications. The anti-CAMP, rabbit polyclonal antibody (Sigma Aldrich HPA029874) was used in the first incubation and it was revealed with streptavidin-Alexa Fluor 555 conjugate (Invitrogen). In the second round, tissues were incubated with α-cytokeratin (peptide 18) FITC conjugated (Sigma Aldrich F4772) diluted 1:100. Nuclei were counterstained with Hoechst, washed with Milli-Q water, and slides were mounted with Fluoromount™ Aqueous Mounting Medium (Sigma Aldrich).
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