Acetone pa

Ac-Pro-His-Cys-Lys-Arg-Met-OH
(AcPHCKRM,
98%) was obtained from Peptide 2.0 Inc. (Chantilly, Virginia, USA).
Acetone p.a. was purchased from Merck (Darmstadt, Germany) and olive
oil from Apoteket AB (Gothenburg, Sweden). [Methyl-³H]thymidine
(2.0 Ci/mmol) was obtained from Perkin-Elmer Biosciences (Waltham
MA, USA). All starting materials and reagents were obtained from commercial
suppliers and were used without prior purification. Tetrahydrofuran
(THF) and CH₂Cl₂ used in reactions with anhydrous
conditions were distilled from Na and CaH₂, respectively.
All reactions were monitored by thin-layer chromatography (TLC) on
silica-plated aluminum sheets (Silica gel 60 F254, E. Merck). Spots
were detected by UV light (254 or 365 nm), or developed with heating,
or anisaldehyde or potassium permanganate staining. All reactions
were carried out using magnetic stirring under an ambient atmosphere
if not otherwise noted. Microwave reactions were carried out using
a Biotage Initiator Sixty in 10–20 mL capped microwave vials
with fixed hold time, normal or high absorption, and 10–30
s pre-stirring. Purification by flash column chromatography was performed
using Merck silica gel Geduran Si 60 (0.063–0.200 mm) or by
using an automatic Biotage SP4 Flash⁺ instrument with pre-packed
columns (surface area 500 m²/g, porosity 60 Å, particle
size 40–63 μm).

Acetone P.A 99.8%, chloroform P.A. 99.8%, dichloromethane P.A. 99.8% (DCM), dimethylformamide P.A 99.5%, dimethyl sulfoxide P.A 99.7%, ethanol P.A. 99.8% (EtOH), tetrahydrofuran P.A. 99.8% (THF), and toluene P.A. 99.0% were purchased from Merck (Rio de Janeiro, Brazil). The photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA) CAS:24650-42-8 was kindly donated by IGM resins (Valinhos, Brazil). Iron (III) chloride hexahydrate (FeCl₃∙6H₂O), iron (II) chloride tetrahydrate (FeCl∙4H₂O), and ammonium hydroxide (NH₄OH) were purchased from Vetec (Duque de caxias, Brazil). Folic acid (FA) 98%, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC), N-Hydroxysuccinimide (NHS), methotrexate hydrate (MTX), fluorescein isothiocyanate (FITC), and enzyme Bromelain were purchased from Sigma-Aldrich (Cotia, Brazil). Cysteine hydrochloride 99.8% (Cys) was purchased from Gemini (Anápolis, Brazil). Novozym 435 (commercial lipase B from Candida Antarctica immobilized on cross-linked polyacrylate beads) was kindly donated by Novozymes, Brazil, A/S. Globalide (Gl) was a kind gift from Symrise (Cotia, Brazil), while ε-caprolactone (CL) was purchased from Sigma-Aldrich (Cotia, Brazil). Globalide and ε-caprolactone were dried under vacuum for 24 h and kept in a desiccator over silica and 4 Å molecular sieves. Water was purified by a Milli-Q water purification system.

Immunohistochemistry was based on the method reported by Morgado and collaborators[20 (link)]. Briefly, spleen fragments frozen in OCT resin (Sakura) were cut into 3–5 μm-thick sections and mounted onto microscope slides (silanized slides; DakoCytomation, Carpinteria, CA, USA). The slides were fixed in acetone PA (Merck, Darmstadt, Germany) and subjected to hydration, endogenous peroxidase blockage (peroxidase blocking reagent; Dako) and nonspecific staining blockage (0.4% BSA; Sigma, USA). The specimens were then incubated with primary antibodies directed against CD3⁺, CD4⁺, CD8⁺, CD21⁺, IFN-γ⁺, IL-10⁺ (Serotec), Ki-67⁺ (eBioscience), laminin, fibronectin, ADAM-10 (Abcam) or MMP-9 (Serotec), followed by incubation with the Labelled Polymer (Envision System-HRP, Dako). Aminoethyl carbazole (AEC kit; Invitrogen) was used as the substrate-chromogen system, and the slides were counterstained with Mayer’s hematoxylin (Sigma). The slides were examined under a light microscope (Zeiss), and the number of marked cells/mm² tissue in the red pulp was determined. Two blinded readers evaluated the slides. Laminin and fibronectin deposits were quantified using ImageJ 1.48v software (NIH, USA), and the results are presented as the percentage of the marked area. Primary antibody suppression was used as the negative control reaction.