After sacrifice, ears were harvested and fixed in neutral buffered formalin (NBF) (Sigma) for 48 h. Samples then underwent tissue processing using a Leica TP1020 automatic tissue processing machine. Tissue was then embedded in paraffin wax blocks (all in the same orientation) and sections were cut with Leica Automated Rotary Microtome (RM 2255) at 8 µm sections and transferred onto slides. The mid-point of each injury was used for analysis (represented by the dashed black bar in the Figures), and the original margins of the injury was demonstrated by the parallel black bars. Slides were imaged using a Plan-Apochromat objective (10x/0.8 M27) on an Axio Scan.Z1 Slide Scanner microscope (Carl Zeiss). Images were compiled and analyzed with Zeiss Zen software.
Axio scan z1 slide scanner microscope
Manufactured by Zeiss
Sourced in Germany
The Axio Scan.Z1 Slide Scanner microscope is a high-performance digital microscope designed for automated scanning and imaging of entire microscope slides. It is capable of capturing high-resolution, high-quality digital images of samples at various magnification levels.
Automatically generated - may contain errors
Lab products found in correlation
Citrisolv, by Thermo Fisher Scientific (3 mentions)
Sodium citrate, by Merck Group (2 mentions)
Zen blue software, by Zeiss (2 mentions)
Tp1020, by Leica (1 mentions)
Automated rotary microtome rm 2255, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Vascular endothelial growth factor (vegf), by Thermo Fisher Scientific (1 mentions)
Everbrite hardset mounting medium with dapi, by Biotium (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Af555, by Thermo Fisher Scientific (1 mentions)
Ab150106, by Abcam (1 mentions)
Ab2072, by Abcam (1 mentions)
Ab53554, by Abcam (1 mentions)
Ab126649, by Abcam (1 mentions)
Lsm 780 confocal microscope, by Zeiss (1 mentions)
Moab 2, by Abcam (1 mentions)
Mkb 2213, by Vector Laboratories (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Merck Group (1 mentions)
Caspase 3, by R&D Systems (1 mentions)
10 protocols using axio scan z1 slide scanner microscope
1
Histological Analysis of Ear Injury
2
Immunofluorescence Staining of Tissue Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Slides were stained with hematoxylin and eosin (H&E) and by immunofluorescence (IF). For IF, sections (7 μm) were deparaffinized in CitriSolv (Fisher Scientific; Fairlawn, NJ, USA) and rehydrated through a series of graded ethanol to distilled water steps. Antigen retrieval (10 mM sodium citrate, pH 6.0, Sigma-Aldrich; St. Louis, Missouri, USA) and blocking (1:500 dilution; 100 μL goat serum: 50 mL TRIS-buffered saline, 0.1% Tween 20 (TBST) for 1 h), steps were performed prior to going through sequential wash (TBST) and the application of primary antibody. Primary antibodies conjugated to fluorophores included: CD14-FITC (Macrophage), CD68-PE (Macrophage), CD90-Alexa488 (MPC) or CD271-Alexa568 (MPC) (all BD Biosciences; Franklin Lakes, NJ, USA), and all slides were counterstained with the nucleic acid stain DAPI (4′,6-diamidino-2-phenylindole) (Sigma-Aldrich; St. Louis, MS, USA) and mounted using FluorSave reagent (Calbiochem; Darmstadt, Germany). Isotype controls for fluorescein isothiocyanate (FITC) and phycoerythrin (PE) demonstrated little to no reactivity (Figure S5 ).
Imaging: Slides were imaged using a Plan-Apochromat objective (20×/0.8 M27) on an Axio Scan.Z1 Slide Scanner microscope (Carl Zeiss; Oberkochen, Germany); DAPI (excitation 353 nm, emission 465 nm), Alexa488 (excitation 493 nm, emission 517 nm), and Alexa568 (excitation 565 nm, emission 576 nm).
Imaging: Slides were imaged using a Plan-Apochromat objective (20×/0.8 M27) on an Axio Scan.Z1 Slide Scanner microscope (Carl Zeiss; Oberkochen, Germany); DAPI (excitation 353 nm, emission 465 nm), Alexa488 (excitation 493 nm, emission 517 nm), and Alexa568 (excitation 565 nm, emission 576 nm).
3
Immunofluorescence Analysis of Tissue Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue sections on slides were also processed for immunofluorescence. Primary antibodies conjugated to fluorophores included anti-Sca1 (clone D7), anti-CD140a (clone APA5) (MSC markers), anti-F4/80 (clone BM8), anti-CD38 (clone 90), anti-CD206 (clone MR6F3) (macrophage), anti-Ki67 (clone SolA15) (proliferation) (all Thermo Fisher Scientific), and anti-collagen I (clone 8-3A5) and II (clone CIIC1) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA). All slides were counterstained with the nucleic acid stain 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) and mounted using FluorSave reagent (Calbiochem, Darmstadt, Germany). Isotype controls for Alexa Fluor 488, 568 or 647 demonstrated little to no reactivity. Slides were imaged using a Plan-Apochromat objective (20×20×/0.8 M27) on an Axio Scan.Z1 Slide Scanner microscope (Carl Zeiss, Oberkochen, Germany).
4
Immunofluorescence Staining of Paraffin Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Paraffin embedded sections (10 µm) were deparaffinized in CitriSolv (Fisher Scientific; Fairlawn, NJ) and rehydrated through a series of graded ethanol to distilled water steps. Antigen retrieval (10 mM sodium citrate, pH 6.0) and blocking (1:500 dilution; 100 µL rat serum:50 mL TRIS-buffered saline, 0.1% Tween 20 (TBST) for 1 h) was undertaken prior to sequential washes (TBST) and primary antibody incubation. Primary antibodies (VEGF [clone # VG1 – Invitrogen Cat# MA5-12184, used at 1:100], CD31 [clone #390 – Invitrogen Cat# 14-0311-82, used at 1:100], Col2 [clone # II-II6B3 – DSHB, Iowa, used at 1:50], αSMA [clone # 1A4 – ABCAM Cat# ab7817, used at 1:100] and PRG4 [clone # 9G3 – Millipore, Cat# MABT401, used at 1:100]) were directly conjugated to fluorophores using the DyLight® 488, 550 or 650 Conjugation Kit [ABCAM]. All slides were mounted using EverBrite™ Hardset Mounting Medium with DAPI (Biotium) for nuclear counterstaining and coverslipped. Slides were imaged using a Plan-Apochromat objective (10x/0.8 M27) on an Axio Scan.Z1 Slide Scanner microscope (Carl Zeiss); DAPI (353 nm/465 nm), EGFP/FITC/Alexa Fluor 488 (493 nm/517 nm), R-PE (565 nm/576 nm), and APC (650 nm/660 nm) specific filters were employed. Isotype controls for Alexa Fluor 488, 550 or 650 demonstrated little to no reactivity.
5
Immunocytochemistry of Hippocampal Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hippocampal cultures were fixed at 3, 5, 7 and 10 days in vitro (DIV) with 4% paraformaldehyde (PFA) for 15 min at room temperature before permeabilization with 0.1% Triton X-100 in PBS for 5 min. Cells were then blocked in 2% FBS in PBS. Incubation with primary antibodies was overnight at 4°C: mouse anti-ankyrin G (1:500, Abcam), rabbit anti-Tau (1:40,000, rPeptide), chicken anti-β3 tubulin (1:300, Merck). Prior to addition of the secondary antibodies, coverslips were washed 5 times with PBS. Coverslips were then incubated with secondary antibodies for 1 h at room temperature. All secondary antibodies were diluted to a concentration of 1:500 (ThermoFisher): donkey anti-mouse Alexa 488, donkey anti-rabbit Alexa 555, and goat anti-chicken Alexa 647. Primary and secondary antibodies were diluted in the blocking solution. After secondary antibody incubation, coverslips were washed 5 times with PBS and DAPI (1:1000) was added for 30 min at RT. Coverslips were then mounted on glass slides using DAKO mounting media and imaged using a 20× air objective on an Axio Scan Z1 slide scanner microscope (Zeiss) and a 60× oil objective on a BX51 microscope (Olympus).
6
Multimodal Immunolabeling of Mouse Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse brain sections were blocked with MOM solution (#MKB-2213, Vector Labs,) and then incubated overnight with primary antibodies against INFγ (1:100, #MCA1301, BioRad), GFAP (1:1000, #ab53554, Abcam) and APP (1:200, #ab2072, Abcam) or against Aβ (MOAB-2 1:1000, #ab126649, Abcam). The next day, the sections were incubated with donkey anti-mouse (AF555, 1:500, #A3157, Thermo Fisher), donkey anti-goat (AF488, 1:500, #A11055, Thermo Fisher) and donkey anti-rabbit (AF647, 1:500, #A31573, Thermo Fisher) or donkey anti-mouse (AF555, 1:500, #ab150106, Abcam) secondary antibodies followed by DAPI staining and Mowiol mounting.
Sections stained with secondary antibodies only were used as negative controls. Additionally, bleed through control stainings were incubated with all three primary antibodies and one secondary separately. Stained mouse brain sections were imaged using a 10x objective on AxioScan.Z1 slide scanner microscope (Zeiss, Oberkochen). To better visualize the distribution of specific markers the slides were imaged also with a 63x oil-immersion objective on a LSM780 confocal microscope (Zeiss, Oberkochen).
Sections stained with secondary antibodies only were used as negative controls. Additionally, bleed through control stainings were incubated with all three primary antibodies and one secondary separately. Stained mouse brain sections were imaged using a 10x objective on AxioScan.Z1 slide scanner microscope (Zeiss, Oberkochen). To better visualize the distribution of specific markers the slides were imaged also with a 63x oil-immersion objective on a LSM780 confocal microscope (Zeiss, Oberkochen).
7
Multi-Fluorescent Imaging Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Slides were imaged using a Plan-Apochromat objective (20×/0.8 M27) on an Axio Scan.Z1 Slide Scanner microscope (Carl Zeiss); DAPI (353 nm/465 nm), FITC (493 nm/517 nm), and phycoerythrin (PE) (565 nm/576 nm).
8
Immunofluorescence Imaging of Synovial and Joint Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human synovial or whole rat and mouse joint sections (7 μm) were deparaffinized in CitriSolv (Fisher Scientific; Fairlawn, NJ, USA) and rehydrated through a series of graded ethanol to distilled water steps. Antigen retrieval (10 mM sodium citrate, pH 6.0, Sigma-Aldrich; St. Louis, Missouri, USA) and blocking (1:500 dilution; 100 μl goat serum: 50 ml TRIS-buffered saline, 0.1% Tween 20 (TBST) for 1 h), steps were performed prior to going through sequential wash (TBST) and the application of primary antibody. Primary antibodies conjugated to fluorophores included: Aggrecan (conjugated to AF 488 or 568), Human/Rat CD90 (BD Biosciences, (Cat# 550402)) or Caspase-3 (R&D, Cat# AF835). All slides were counterstained with the nucleic acid stain DAPI (4’,6-diamidino-2-phenylindole) (Sigma-Aldrich; St. Louis, Missouri, USA) and mounted using FluorSave reagent (Calbiochem; Darmstadt, Germany). Isotype controls for AF 488 or AF 568 demonstrated little to no reactivity. Slides were imaged using a Plan-Apochromat objective (×20/0.8 M27) on an Axio Scan.Z1 Slide Scanner microscope (Carl Zeiss; Oberkochen, Germany); DAPI (excitation 353 nm, emission 465 nm), Alexa488 (excitation 493 nm, emission 517 nm), and Alexa568 (excitation 565 nm, emission 576 nm).
9
Immunofluorescence Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence (IF) staining was performed as described previously (23) . Briefly, cells or tissues were stained for the indicated proteins, and nucleus were labeled with DAPI. Primary and secondary antibodies are listed in Supplementary Table S2. Slides were scanned with an axioscan Z1 slide scanner microscope (Zeiss) using a 20Â/ 0.75NA objective. Images were analyzed using Zen blue software (Zeiss) and Qupath.
10
Immunofluorescence Staining Protocol for Tissue Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IF staining was performed as previously described (32) (link). Briefly, tissues were stained for p-eIF4E, p-MNK1, IL-33, α-smooth muscle actin (α-SMA), CD45 and CD8, and nucleus were labeled with DAPI. Primary and secondary antibodies were listed in Supplementary Table 2. Slides were scanned with an axioscan Z1 slide scanner microscope (Zeiss) using a 20x/0.75NA objective.
Images were analyzed using Zen blue software (Zeiss) and Qupath. A detailed description of analysis procedure can be found in supplemental methods.
Images were analyzed using Zen blue software (Zeiss) and Qupath. A detailed description of analysis procedure can be found in supplemental methods.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!