The complexity of human astrocytes stays poorly outlined in major human tissue, requiring higher instruments for his or her isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be utilized to efficiently isolate and examine human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding issues related to dealing with recent tissue. Nonetheless, efforts to equally isolate astroglia from the non-neuronal (NeuN-) ingredient are missing.
A lately developed and validated immunotagging technique makes use of three transcription issue antibodies to concurrently isolate enriched neuronal (NeuN+), astrocyte (paired field protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, recent (unfixed) snap-frozen postmortem human temporal neocortex tissue.
This system was proven to be helpful for the characterization of cell type-specific transcriptome alterations in major pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and seize astrocytes in each resting and reactive circumstances.
This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, together with tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating methods and high quality management metrics for optimizing sensitivity and specificity throughout sorting and for confirming astrocyte enrichment; and really helpful procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn decision. This protocol is relevant for non-necrotic, fresh-frozen, human cortical specimens with numerous pathologies and really helpful postmortem tissue assortment inside 24 h.

Principal Function of Antibodies in Demyelination and Axonal Harm in A number of Sclerosis

Antibodies and oxidative stress are hallmarks of a number of sclerosis (MS) lesions. We aimed to make clear the relation between them, their position in MS sufferers and to research their specificity, evaluating MS with classical neurodegenerative ailments (ND). Mind samples from 14 MS instances, 6 with ND and 9 controls (with out neurological ailments). Immunohistochemistry assays have been used to detect oxidized lipids (EO6), IgG and IgM, oligodendrocytes (Olig2), axons (NF, neurofilament) and mobile (TUNEL) and axonal harm (APP, amyloid precursor protein).
We didn’t observe EO6 in controls. All samples from MS sufferers confirmed EO6 in oligodendrocytes and axons inside lesions. We didn’t detect co-localization between EO6 and antibodies. Neither did we between EO6 and TUNEL or APP. 94.4% of TUNEL-positive cells in regular showing white matter have been additionally stained for IgG and 75.5% for IgM. IgM, however not IgG, co-localized with APP. EO6 was related to axonal harm in amyotrophic lateral sclerosis (ALS).
We didn’t observe affiliation between antibodies and mobile or axonal harm in ND sufferers. MS sufferers confirmed a better variety of B cells and plasma cells within the lesions and meninges than controls. The variety of B cells and plasma cells was related to the presence of antibodies and with the exercise of the lesions. We noticed a predominant position of B lymphocytes within the improvement of MS lesions. Antibodies contribute to the oligodendrocyte and axonal harm in MS. Oxidative stress was related to axonal harm in ALS.

Correlation Between Immunohistochemistry and Sequencing in H3G34-Mutant Gliomas

Recurrent glycine-to-arginine/valine alterations at codon 34 (G34R/V) inside H3F3A gene characterize a subset of hemispheric high-grade gliomas (HGG) affecting kids and younger adults. These tumors, outlined as G34R/V-mutant gliomas, are histologically heterogenous, with microscopic options of both HGG or embryonal tumors (primitve neuroectodermal tumor-like options).
To evaluate the worth of immunohistochemistry (IHC) to detect G34R/V-mutated instances, we examined anti-histone G34V (clone 329E5) and anti-histone G34R (clone RM240) antibodies in a sequence of 28 formalin-fixed and paraffin-embedded samples. A complete of 28 instances of hemispheric, IDH-wt HGG primarily affecting kids and younger adults have been evaluated by IHC and by sequencing. The median age of sufferers at analysis was 17 years (0.1 to 26 y). By IHC, 10 of the 28 instances confirmed nuclear positivity for G34R and three of the 28 instances for G34V.
Molecular evaluation of G34R/V-mutation standing was profitable in 24 of the 28 instances. Mutation at glycine 34 of the H3F3A gene was recognized in 9 of the 24 tumors (37%) by direct sequencing, revealing 7 of 9 constructive case by sequencing and a pair of of 9 false destructive instances by IHC.
Two of 15 destructive case by sequencing demonstrated a false positivity by IHC. In whole, in 4 (16.6%) of 24 instances, IHC and mutational outcomes have been discordant: 2 tumors have been destructive by IHC (false destructive) however harbored G34R mutation by sequencing, and a pair of instances have been constructive by IHC (false constructive by IHC) however wild kind by sequencing.
Furthermore, most mutated instances confirmed lack of ATRX expression and/or p53 expression. The positivity by IHC with particular antibody examined just isn’t extremely predictive for presence of G34R/V mutation, however affirmation by sequencing is necessary; G34R/V mutations ought to be suspected in all hemispheric tumor IDH1/2 wild kind, exhibiting lack of OLIG2 and ATRX and/or p53 expression.

Oligodendrocyte Lineage Marker Expression in eGFP-GFAP Transgenic Mice

Oligodendrocytes, the myelinating cells of the central nervous system, orchestrate a number of key mobile capabilities within the mind and spinal wire, together with axon insulation, power switch to neurons, and, ultimately, modulation of immune responses. There’s rising curiosity for acquiring dependable markers that may particularly label oligodendroglia and their progeny.
In lots of research, anti-CC1 antibodies, presumably recognizing the protein adenomatous polyposis coli (APC), are used to label mature, myelinating oligodendrocytes. Nonetheless, it has been mentioned whether or not anti-CC1 antibodies may acknowledge as effectively, beneath pathological circumstances, different cell populations, significantly astrocytes.

Olig2 Antibody

48708-50ul 50ul
EUR 239

OLIG2 Antibody

45197-100ul 100ul
EUR 252

OLIG2 Antibody

45197-50ul 50ul
EUR 187

OLIG2 Antibody

DF8004 200ul
EUR 304
Description: OLIG2 Antibody detects endogenous levels of total OLIG2.

OLIG2 Antibody

1-CSB-PA016329GA01HU
  • EUR 597.00
  • EUR 333.00
  • 150ul
  • 50ul
  • Form: Liquid
  • Buffer: PBS with 0.02% Sodium Azide, 50% Glycerol, pH 7.3. -20℃, Avoid freeze / thaw cycles. Antigen Affinity purified
Description: A polyclonal antibody against OLIG2. Recognizes OLIG2 from Human, Mouse, Rat. This antibody is Unconjugated. Tested in the following application: ELISA, WB, IHC

olig2 Antibody

1-CSB-PA24549A0Rb
  • EUR 317.00
  • EUR 335.00
  • 100ug
  • 50ug
  • Form: Liquid
  • Buffer: Preservative: 0.03% Proclin 300
    Constituents: 50% Glycerol, 0.01M PBS, pH 7.4 >95%, Protein G purified
Description: A polyclonal antibody against olig2. Recognizes olig2 from Zebrafish. This antibody is Unconjugated. Tested in the following application: ELISA, WB; Recommended dilution: WB:1:500-1:5000

OLIG2 Antibody

ABD8004 100 ug
EUR 438

Olig2

GT15132 100 ug
EUR 526

Olig2

RA25081 100 ul
EUR 565

Olig2 Conjugated Antibody

C48708 100ul
EUR 397

Polyclonal OLIG2 Antibody

AMM06894G 0.1 mg
EUR 659
Description: A polyclonal antibody raised in Rabbit that recognizes and binds to Human OLIG2 . This antibody is tested and proven to work in the following applications:

Monoclonal OLIG2 Antibody

AMM06895G 0.1ml
EUR 484
Description: A Monoclonal antibody against Human OLIG2. The antibodies are raised in Mouse. This antibody is applicable in WB

Polyclonal Olig2 Antibody

AMM06896G 0.1ml
EUR 528
Description: A polyclonal antibody raised in Rabbit that recognizes and binds to Human Olig2 . This antibody is tested and proven to work in the following applications:

OLIG2 Conjugated Antibody

C45197 100ul
EUR 397

Anti-OLIG2 Antibody

A1656-100
EUR 479

anti- OLIG2 antibody

FNab05986 100µg
EUR 505.25
  • Recommended dilution: WB: 1:500 - 1:1000
  • IHC: 1:500 - 1:1500
  • IP : 1:500 - 1:1000
  • Immunogen: oligodendrocyte lineage transcription factor 2
  • Uniprot ID: Q13516
  • Gene ID: 10215
  • Research Area: Epigenetics, Metabolism, Cancer, Developmental biology, Neur
  • Show more
Description: Antibody raised against OLIG2

anti- OLIG2 antibody

FNab05987 100µg
EUR 548.75
  • Immunogen: oligodendrocyte lineage transcription factor 2
  • Uniprot ID: Q13516
  • Gene ID: 10215
  • Research Area: Epigenetics, Metabolism, Cancer, Developmental biology, Neuroscience, Stem Cells
Description: Antibody raised against OLIG2

Anti-OLIG2 antibody

PAab05986 100 ug
EUR 355
On this examine, we used transgenic mice by which astrocytes are labeled by the improved inexperienced fluorescent protein (eGFP) beneath the management of the human glial fibrillary acidic protein (GFAP) promoter. By detailed co-localization research we have been in a position to reveal {that a} important proportion of eGFP-expressing cells co-express markers of the oligodendrocyte lineage, such because the transcription issue Oligodendrocyte Transcription Issue 2 (OLIG2); the NG2 proteoglycan, also referred to as chrondroitin sulfate proteoglycan 4 (CSPG4); or APC. The present discovering that the GFAP promoter drives transgene expression in cells of the oligodendrocyte lineage ought to be thought-about when deciphering outcomes from co-localization research.

LEAVE A REPLY

Please enter your comment!
Please enter your name here